Materials and methods relating to the treatment of leukaemias

ABSTRACT

The invention provides materials and methods capable of modulating the strong-self-association of chimeric transcription factors to form high molecular weight (HMW) complexes. The invention further provides compounds comprising the oligomerization domains of oligomeric substances and a polypeptide for modulating the activity of that polypeptide intra or inter-cellularly.

FIELD OF THE INVENTION

[0001] The present invention relates to the materials and methodsinvolved in the treatment of leukaemias. Particularly, but notexclusively, the present invention relates to materials and methodscapable of modulating the strong self-association of chimerictranscription factors to form high molecular weight (HMW) complexes ascompared to the naturally occurring monomeric transcription factor. Thepresent invention is primarily concerned with those transcriptionfactors involved in differentiation of primary hematopoietic precursors.

BACKGROUND OF THE INVENTION

[0002] Acute myeloid leukaemias (AMLs) are characterised by chromosomaltranslocations resulting in the generation of chimeric genes and fusionproteins (Look, 1997; Rabbitts, 1994; Rabbitts, 1991; Tenen et al.,1997). Ectopic expression of fusion proteins induces differentiationblock of hemopoietic precursors and leukemias in animal models (Du etal., 1999; Gelmetti et al., 1998; Grignani et al., 1993; Grignani etal., 1996; Lavau et al., 1997; Pereira et al., 1998; Ruthardt et al.,1997; Schwaller et al., 1998; Slanyet al., 1998; Brown et al., 1997;Grisolano et al., 1997; Westervelt and Ley, 1999). One of the genesinvolved in the AML-associated translocations encodes almost invariablyfor a transcription factor, which is physiologically involved inhematopoietic differentiation (such as retinoic acid receptor α—RARAα—inacute promyelocytic leukaemia—APL—, or AML-1 in acute myelogenousleukaemia: Look, 1997; Rabbitts, 1994; Rabbitts, 1991; Shivdasani andOrkin, 1996; Tenen et al., 1997). According to the current model ofleukaemogenesis, the differentiation block is the consequence of thealtered transcriptional properties of these chimeric transcriptionfactors (Look, 1997; Shivdasani and Orkin, 1996; Tenen et al., 1997).The molecular mechanisms of the oncogenic conversion, however, arelargely unknown.

[0003] Recent findings demonstrated that aberrant recruitment of thenuclear corepressor (NCoR)-histone deacetylase (HDAC) complex is crucialto the activation of the leukemogenic potential of RAR and AML 1 in thefusion proteins PML-RAR and AML 1-ETO (Cheng et al., 1999; David et al.,1998; Gelmetti et al., 1998; Grignani et al., 1998; Guidez et al., 1998;He et al., 1998; Lutterbach et al., 1998; Wang et al., 1998). Histoneacetylation levels influence chromatin structure in a manner tightlylinked to transcriptional activity: high levels of histone acetylationare observed at the promoters of transcribed genes, whereashypo-acetylation has been correlated to silenced genes (Grunstein, 1997;Pazin and Kadonaga, 1997). It is expected, therefore, that modificationof the chromatin structure at the target promoters of the fusionproteins represents one important mechanism of leukaemogenesis (Minucciand Pelicci, 1999; Redner et al., 1999; Stunnenberg et al., 1999).

[0004] Unliganded RARs repress transcription by recruiting the NCoR/HDACcomplex: RA triggers dissociation of the NCoR/HDAC complex andrecruitment of several co-activators (PCAF, p300/CBP, SRC-1) endowedwith histone acetylase activity , thus leading to transcriptionalactivation (Chambon, 1996; Mangelsdorf and Evans, 1995; Minucci andPelicci, 1999; Wolffe et al., 1997; Xu et al., 1999). AML 1 is atranscriptional activator associated with p300/CBP (Kitabayashi et al.,1998), while ETO interacts with NCoR and recruits HDAC activity in vivo(Gelmetti et al., 1998; Lutterbach et al., 1998; Wang et al., 1998). ThePML-RAR fusion protein retains the NCoR binding site of RAR, whereas AML1-ETO has lost the p300/CBP interaction site of AML 1 but retains theNCoR/HDAC binding site of ETO (Minucci and Pelicci, 1999). In line withthese findings, PML-RAR and AML 1/ETO form stable complexes withNCoR-HDAC (Gelmetti et al., 1998; Grignani et al., 1998; He et al.,1998; Lin et al., 1998; Lutterbach et al., 1998). Mutation of the NCoRbinding site(s) impairs the biological activity of the two fusionproteins, indicating that formation of aberrant complexes withhistone-modifying enzymes is essential for leukaemogenesis (Gelmetti etal., 1998; Grignani et al., 1998).

[0005] The mechanisms leading to abnormal recruitment of the NCoR/HDACcomplex differ in the case of PML-RAR or AML 1-ETO. In AML 1-ETO, lossof p300/CBP and gain of NCoR association might be sufficient to endowthe fusion protein with constitutive transcriptional repressiveactivity. In contrast, PML-RAR has the same property of RAR to recruitNCoR in the unliganded state: how the association with NCoR becomesabnormal when RAR is fused to PML remains unclear.

SUMMARY OF THE INVENTION

[0006] The present inventors have for the first time established thatthe formation of HMW complexes of chimeric transcription factors(PML-RAR and AML1-ETO) results in abnormal recruitment of the NCoR/HDACcomplex. This discovery has provided an important insight intomechanisms leading to the production of HMW complexes of chimerictranscription factors, e.g. PML-RAR and AML1-ETO, and as a result theabnormal recruitment of the NCoR-HDAC. This knowledge would have anumber of important and industrially applicable implications,particularly as regards the treatment or diagnosis of leukaemias.Further, an understanding of the mechanisms involved in the formation ofHMW complexes of oligomeric factors leading to their altered activity,opens the way to identifying a domain within other naturally occurringoligomeric factors e.g. chimeric transcription factors or other classesof proteins. This domain may then be used as a tool to enhance, throughself-association, the functional properties of a given protein, notalready present in nature as a strongly self-associating factor. Inother words, manipulation of this domain may be equivalent (for thefunction of a protein) to genetically manipulating a promoter for a genethat normally (unmanipulated) has a weak promoter, to make it stronger.A further extension of the studies has also led the inventors to theconclusion that use of said domain to promote self-association,especially in the case of proteins already present as oligomericcomplexes in nature, may lead through an oligomerization chain reactionto a reduced activity of a given protein.

[0007] The inventors have found that PML-RAR (unlike RAR) forms tightlyinteracting oligomers in vivo and that the coiled coil region of PML isthe structural determinant for strong self-association andoligomerization. They have been able to show that oligomerization isresponsible, per se, for (a) the increased recruitment of NCoR, (b)constitutive transcriptional repressive activity on RA-target promoters;and (c) leukemogenic potential of the fusion protein. A similarpotential to form oligomeric structures has also been observed for theother APL-associated (PLZF-RAR and NPM-RAR) fusion proteins. AML1-ETOwas also found in HMW complexes and shown to form oligomeric complexes,owing to the ETO moiety of the fusion protein. A derivative of AML 1-ETOdevoided of the capacity to form HMW complexes showed a decreasedcapacity to interact with NCoR, impaired transcriptional repressiveactivity and was unable to block terminal differentiation ofhematopoietic precursors. These findings highlight the physical statusof a transcription factor as a potent mechanism to modulate its abilityto recruit co-regulators, and indicate thatself-association/oligomerization by heterologous interaction interfacesis a novel mechanism for the oncogenic conversion of a transcriptionfactor in leukaemias. Thus, having for the first time determined thismechanism, the inventors have realised that disruption or inhibition ofthe formation of unwarranted oligomeric complexes provides a target fortherapeutical intervention in the treatment of this disease. Theinformation provided herein allows for the provision of materials andmethods for (i) affecting the biological pathway involved indifferentiation of primary hematopoietic precursors; (ii) affecting thebiological pathway(s) involved in oncogenic transformation by alteredtranscription factors; (iii) assessing the presence of transcriptionfactors with the above-mentioned altered properties in cancer samples;(iv) modifying the activity of a given protein by fusion with theheterologous coiled coil domain from PML with the intent of enhancingits functional activity or to reduce its functional activity; (v)modifying the activity of a given protein by fusion with theheterologous coiled coil domain from PML with the intent of reducing itsfunctional activity.

[0008] Thus, in summary, a specific domain (termed hereinafter“oligomerization domain”) within transcription translocation proteins(e.g. PML-RARα), implicit in the cause of leukaemias represents thenecessary means by which these proteins self associate with each other.This is known to occur prior to binding to the DNA where the fusionprotein inhibits DNA transcription and thereby brings about thephenotypic changes manifested in leukaemic patients e.g. loss ofdifferentiation. The inventors have shown for the first time that lossof this oligomerization domain and concomitant loss of the ability toform self-associating homodimers is sufficient to render these mutatedproteins harmless and restore the cancerous cells back to their normaldifferentiated state. Until now it has never been shown that theformation of these oligomers with the enhanced capacity to recruit NcoRis a prerequisite for disease progression. Surprisingly, if the diseasedtranslocation proteins remain as monomeric or single units they have nodebilitating effects and normal cell differentiation occurs.

[0009] Therefore, at its most general, the present invention providesmaterials and methods which detect or affect the formation of tightlyself-interacting oligomeric complexes. In particular, the invention isconcerned with oligomeric complexes of chimeric factors, e.g.transcription factors.

[0010] A chimeric transcription factor is a fusion protein comprising atranscription factor or part thereof and a second protein—that may—ormay not—be a transcription factor itself. The fusion protein is encodedby a gene altered as a result of a translocation event. These chimerictranscription factors have altered activity with respect to the wildtype transcription factor. Examples of chimeric transcription factorsinclude PML-RAR and AML1-ETO.

[0011] Preferably the chimeric transcription factors are products of thechromosomal translocations associated with leukaemias. Even morepreferably the chimeric transcription factors are PML-RAR and AML1-ETO.

[0012] Oligomerization (trimers or hexamers in PML-RAR's case, but couldbe dimers with a different “n” oligomerization number) is critical toleukaemogenesis due to the increased concentration of binding sites forco-regulatory factors including NCoR which binds HDAC. HDAC has beenshown to inhibit transcription. Thus, owing to the increased localconcentration of NCoR and/or because of increased stability of NCoRbinding (since as soon as one molecule disassociates there is anotherbinding site very close by to which it can bind), the addition of RA atnatural concentrations is no longer sufficient enough to replace theNCoR/HDAC and allow transcription to proceed. Moreover, oligomerizationof PML-RAR and AML1-ETO transcription factors, through the aviditycomponent (owing to multimerization of the NCoR binding sites) andthrough entropic effects (owing to an increase in the localconcentration of NCoR binding sites), leads to a dramatic increase inthe stability of their interaction with transcriptional co-repressorsand possibly other co-regulators, thus leading to deregulatedtranscription.

[0013] This oligomerization principle is shown herein in the fusionproteins PML-RAR and AML1-ETO but is likely to be true in otherleukaemia associated translocation proteins involving transcriptionfactors and therefore in any number of diseases where translocationproteins are involved. For convenience, the text concentrates onchimeric transcription factors as the oligomeric factors. However, theskilled person will appreciate that the aspects of the invention may beapplied to other oligomeric factors, e.g. TNF, p53 etc. In the contextof this invention, an oligomeric factor is a polypeptide including achimeric or fusion polypeptide that is capable of binding to otheroligomeric factors to form an oligomeric complex. A monomeric factor isa polypeptide that exists as a single entity and does not naturally formcomplexes, e.g. thyroid receptor.

[0014] Thus, in a first aspect of the present invention there isprovided a method of determining the presence or absence of a HighMolecular Weight (HMW) complex comprising a chimeric transcriptionfactor, preferably PML-RAR or AML 1-ETO, comprising the steps ofobtaining a biological sample from a patient and detecting the presenceor absence of said HMW complex. A HMW complex comprises two or moreoligomeric factors, e.g. chimeric transcription factors which form atightly self-interacting oligomeric complex. The HMW complex maycomprise dimers, trimers, tetramers, pentamers, hexamers etc, of theoligomeric factors, e.g. chimeric transcription factor.

[0015] The complex may be detected using standard techniques known tothose skilled in the art, such as using a specific binding membercapable of binding to the complex, e.g. an antibody binding domain, thespecific binding member being labelled so that binding of the specificbinding member to the complex is detectable. For example, in the case ofacute promyelocytic leukaemia (APL) chimeric transcription factors(PML-RAR, PLZF-RAR, NPM-RAR and NuMA-RAR), the specific binding membermay be labelled (radioactively, fluorescently etc.) retinoic acid thatbinds the RAR moiety of the chimeric transcription factors.

[0016] It may also be necessary to further determine the molecularweight of the product specifically bound by the specific binding member.This will serve to distinguish between detection of the HMW complex ofinterest and naturally occurring chimeric transcription factors whichhave not formed HMW complexes by, for example oligomerization, or evenwild type non-chimeric transcription factors. The molecular weight ofthe bound product may be determined by, for example, size-exclusionchromatography and subsequent analysis of the column fractions byretinoic acid labelling, immuno-based detection techniques-Western blot,or ELISA.

[0017] Other techniques exist which may be used to indicate theoligomeric state of the HMW complex detected in the biological sample.These include determining stokes radius, or sedimentation coefficientetc. Other techniques will be apparent to the skilled person.

[0018] In order to determine the presence of absence of the HMWcomplexes in a biological sample, a comparison may be made with acontrol sample of known molecular weight of the chimeric transcriptionfactors which have not formed HMW complexes and the wild typenon-chimeric transcription factors.

[0019] The choice of biological sample will depend on the HMW complexbeing determined. For example, if the complex is formed by chimerictranscription factors PML-RAR or AML 1-ETO then the biological samplewould preferably be blood. However, other examples of biological fluidsinclude plasma, serum, tissue sample, tumour samples, saliva and urine.

[0020] This aspect of the invention may be used to diagnose a patientsuspected of having an abnormality in the transcriptional control ofcertain genes due to abnormal chromosomal translocations, leading to thedevelopment of a disease such as cancer, or it may be used to determinethe susceptibility of a patient to a particular form of disease e.g.cancer. For example, one could determine the presence or absence of HMWtranscription factor complexes, reflecting their oligomeric nature. Thisdetermination has the advantage of being able to not only confirm theabnormality but also to determine the exact type of abnormality and, asa consequence, direct the specific treatment of the patient. Forexample, if a patient is suspected of having a form of acute leukaemia,a blood sample may be obtained and tested for the presence or absence ofHMW chimeric transcription factor complexes (e.g. comprising PML-RAR orAML 1-ETO). Should any abnormal HMW chimeric transcription factorcomplexes be found then the patient may, following additionalcytogenetical analysis if necessary, be diagnosed as having, orsusceptible for, acute myeloid leukaemias (AML) or acute promyelocyticleukaemia (APL). In addition, the screening for the presence of HMWchimeric transcription factor complexes might be extended to other formsof cancer, where the detection of such complexes could also represent acritical factor for the therapeutical strategy.

[0021] Thus, an embodiment of this aspect of the present inventionprovides a method of diagnosing an acute myeloid leukaemia or APLcomprising the steps of obtaining a biological sample from a patient,preferably blood or serum, and testing said sample for the presence of aHMW complex comprising PML-RAR or AML 1-ETO.

[0022] In a second aspect of the present invention there is provided amethod of treating a patient having a disease, such as cancer,associated with the formation of HMW complexes comprising chimerictranscription factors thereby resulting in the abnormal transcriptionalcontrol of gene(s), said method comprising administering to said patienta factor capable of disrupting the activity or formation of said HMWcomplexes. Preferably, the factor prevents, disrupts or inhibits theformation of the HMW complex. For example, if the complex was formedthrough the oligomerization of PML-RAR then the factor may be capable ofpreventing or disrupting the oligomerization. The present inventors havediscovered for the first time that in the case of PML-RAR the structuraldeterminant of oligomerization of the chimeric transcription factor, aswell as of the natural PML protein, is the coiled coil region of PML.Thus, an embodiment of this aspect of the present invention would be toblock the activity of this region of the factor in question, e.g. PML,such that oligomerization could not take place.

[0023] This disruption is preferably achieved by the administration offactors such as binding members which are capable of specificallybinding to the coiled coil region of PML such that oligomerizationcannot take place. Examples of such binding members include (I) antibodybinding domains specific for an epitope in the region in question; (ii)oligopeptides comprising the coiled coil domain of PML itself in thecase of PML-RAR (and therefore capable of binding PML-RAR HMW complexesand disrupting them), or the self association domain specific for otherchimeric transcription factor; (iii) small molecules derived fromscreening for compounds exhibiting the capability ofpreventing/disrupting specific HMW complexes (see below).

[0024] In this context, disruption may be taken to mean either theprevention of complex formation or, if the complex has already formed,the prevention of complex activity such as transcriptional repressiveactivity. Prevention of complex activity may be achieved by break-up ofthe complex itself.

[0025] As mentioned above, the present inventors have determined for thefirst time that the formation of these HMW complexes (includingoligomerization) are responsible for (a) the increased recruitment ofNCoR; (b) the localised increase in HDAC concentration; (c) theconstitutive transcriptional repressive activity; and (d) theleukemogenic potential of the fusion protein of the chimerictranscription factors. Thus, the inventors have determined that as aresult of the HMW complex formation, these fusion proteins have anincreased capacity to interact with NCoR.

[0026] As also mentioned above, the present inventors have shown thatfor the PML-RAR fusion protein the structural determinant ofoligomerization (oligomerization domain) of the chimeric transcriptionfactor is the coiled coil region of PML, and that the oligomerizationdomain of AML1-ETO comprehends a coiled coil region. In the case of theother APL fusion proteins, NuMA-RAR, PLZF-RAR, and NPM-RAR, theoligomerization domain contributed by NuMA is a coiled coil region,whereas PLZF and NPM show a different folding of their oligomerizationdomains. The oligomerization domain (or coiled coil) is the structuraldeterminant for strong self-association and oligomerization.

[0027] In PML this coiled coil domain has the following amino acidsequence (SEQ ID NO 1).SELKCDISAEIQQRQEELDAMTQALQALQEQDSAEGAVHAQMHAAVGQLGRARAETEELIRERVRQVVAHVRAQERELLEAVDARYQRDYEEMASRLGRLDAVLQRIRTGSALVQRMKCYASDQEVLDMHGFLRQALCRLR

[0028] The murine coiled coil domain of PML has the following amino acidsequence (SEQ ID NO 2).SHLHCDIGEEIQQWHEELGTMTQTLEEQGRTFDSAHAQMCSAIGQLDHARADIEKQIGARVRQVVDYVQAQERELLEAVNDRYQRDYQEIAGQLSCLEAVLQRIRTSGALVKRMKLYASDQEVLDMHSFLRKALCSLR

[0029] Thus, the present invention further provides assays using apeptide (produced in vitro, or in vivo through methods available to theskilled person) having either the murine or human sequence given above,or a variant thereof to find substances capable of modulating theoligomerization domain so that self-association of the chimerictranscription factors is prevented or reduced. Analogously, the presentinvention further provides assays using a peptide having sequencescorresponding to the oligomerization domain of any given chimerictranscription factor, or a variant thereof, to find substances capableof modulating the oligomerization domain so that self-association of thechimeric transcription factors is prevented or reduced.

[0030] One class of substance that may be used to disrupt theoligomerization domain are peptides based on the sequence motifs of thecoiled coil region which causes oligomerization/strong self-association.Such peptides tend to be small molecules, and may be about 40 aminoacids in length or less, preferably 35 amino acids in length morepreferably 30 amino acids in length, or less, more preferably 25 aminoacids in length or less, more preferably 20 amino acids in length orless, more preferably about 15 amino acids or less, more preferablyabout 10 amino acids or less, or 9, 8, 7, 6 5 or less in length. Thepresent invention also encompasses peptides which are sequence variantsor derivatives of a wild type oligomerization domain, i.e. the coiledcoil domain as given above.

[0031] Preferably, the amino acid sequence shares homology with afragment of the coiled coil domain sequence shown preferably at leastabout 30%, or 40%, or 50%, or 60%, or 70%, or 75%, or 80%, or 85%homology, or at least about 90% or 95% homology. Thus, the coil coileddomain of the chimeric transcription factor may include 1, 2, 3, 4, 5,greater than 5, or greater than 10 amino acid alterations such assubstitutions with respect to the wild-type sequence.

[0032] As is well-understood, homology at the amino acid level isgenerally in terms of amino acid similarity or identity. Similarityallows for “conservative variation”, i.e. substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas arginine for lysine, glutamic for aspartic acid, or glutamine forasparagine. Similarity may be as defined and determined by the TBLASTNprogram, of Altschul et al, J. Mol. Biol., 215:403-10, 1990, which is instandard use in the art. Homology may be over the full-length of therelevant peptide or over a contiguous sequence of about 5, 10, 15, 20,25, 30 or 35 amino acids, compared with the relevant wild-type aminoacid sequence.

[0033] Further, other small molecules or compounds may be used tomodulate the structural determinant of oligomerization such thatself-association of the chimeric transcription factors cannot takeplace. For example, lipids, phospholipids, oligosaccarides etc may beused. These molecules may have the advantage of possibly being easier toproduce and deliver than peptides.

[0034] In one general aspect, the present invention further provides anassay method for a substance with ability to modulate the structuraldeterminant of oligomerization (oligomerization domain) of an oligomericfactor such that strong self-association of the oligomeric factors toform oligomeric complexes is prevented or reduced, the method including:

[0035] (a) bringing into contact a first oligomeric factor or thefunctional self-association part thereof, a second oligomeric factor thefunctional self-association part thereof, and a test compound, underconditions wherein, in the absence of the test compound being aninhibitor of association of said oligomeric factors, said oligomericfactors or functional self-association parts thereof interact or bind;and,

[0036] (b) determining interaction or binding between said oligomericfactors or functional self-association parts thereof.

[0037] It will be apparent to the skilled person that to perform anassay method as defined above, the whole oligomeric factor need not beused. Indeed, it would be sufficient to use that part of the factor thatis involved with the oligomerization/self-association of that factor.Thus, in the case of PML it would be possible to use a peptidecomprising the coiled coil region of this transcription factor or even afragment of this region known to be involved in oligomerization/selfassociation. Any assay developed with an isolated region known to beinvolved in oligomerization/self-association will be subsequentlyextended to the whole transcription factor.

[0038] A test compound which disrupts, reduces, interferes with orwholly or partially abolishes binding or interaction between saidmonomeric chimeric transcription factors, and which may modulate thebioactivity of said transcription factors, may thus be identified.

[0039] Another general aspect of the present invention provides an assaymethod for a test compound able to bind the relevant region of theoligomerization domain (e.g. the coiled coil domain); the methodincluding:

[0040] (a) bringing into contact a substance which includes aoligomerization domain(e.g. the coiled coil domain) which allowsself-association of the oligomeric factors e.g. chimeric transcriptionfactors, or a variant, derivative or analogue thereof, and a testcompound; and,

[0041] (b) determining binding between said oligomerization domain andthe test compound.

[0042] A test compound found to bind to the relevant portion of theoligomerization domain may be tested for ability to disruptself-association of the oligomeric factors under test and/or the abilityto affect the bioactivity or other activity mediated by thetranscription factors.

[0043] Performance of an assay method according to the present inventionmay be followed by isolation and/or manufacture and/or use of acompound, substance or molecule which tests positive for ability tointerfere with the self-association of the oligomeric factors and/ormodulate their bioactivity.

[0044] The precise format of an assay of the invention may be varied bythose of skill in the art using routine skill and knowledge. Forexample, interaction between substances may be studied in vitro bylabelling one with a detectable label and bringing it into contact withthe other which has been immobilised on a solid support. Suitabledetectable labels, especially for petidyl substances include³⁵S-methionine which may be incorporated into recombinantly producedpeptides and polypeptides. Recombinantly produced peptides andpolypeptides may also be expressed as a fusion protein containing anepitope which can be labelled with an antibody.

[0045] The protein which is immobilized on a solid support may beimmobilized using an antibody against that protein bound to a solidsupport or via other technologies which are known per se. A preferred invitro interaction may utilise a fusion protein includingglutathione-S-transferase (GST). This may be immobilized on glutathioneagarose beads. In an in vitro assay format of the type described above atest compound can be assayed by determining its ability to diminish theamount of labelled peptide or polypeptide which binds to the immobilizedGST-fusion polypeptide. This may be determined by fractionating thematerial attached to the glutathione-agarose beads by SDS-polyacrylamidegel electrophoresis. Alternatively, the beads may be rinsed to removeunbound protein and the amount of protein which has bound can bedetermined by counting the amount of label present in, for example, asuitable scintillation counter.

[0046] Alternatively, FRET (Fluorescence Resonance EnergyTransfer)—based assay may be developed that will show when and howstrongly these coiled-coil domains are self associating in vitro i.e. oneasy to handle and high-throughput microwell plates.

[0047] An assay according to the present invention may also take theform of an in vitro assay. The in vivo assay may be performed in a cellline such as a yeast strain or mammalian cell line in which the relevantpolypeptides or peptides are expressed from one or more vectorsintroduced into the cell. In this case, the demonstration of theinteraction—or its prevention, or its disruption by the screenedcompounds—between the self-associating moieties under study will havethe form of measurable enzymatic activity from a reporter enzyme, orfluorescence, or FRET phenomena.

[0048] In a third aspect of the present invention there is provided anassay method for a substance with the ability to disrupt interaction orbinding between NCoR and a HMW complex formed from a chimerictranscription factor, for example, PML-RAR or AML 1-ETO, the methodincluding

[0049] (a) bringing into contact said HMW complex or a variant,derivative, or analogue thereof, including the binding region for NCoR,a substance including the relevant fragment of NCoR or a variant,derivative or analogue thereof, and a test compound, under conditionswherein, in the absence of the test compound being an inhibitor ofinteraction between or binding of said substances, said substances bind;and

[0050] (b) determining the interaction or binding between saidsubstances.

[0051] A test compound which disrupts, reduces, interferes with orwholly or partially abolishes binding or interaction between saidsubstances (e.g. including a NCoR binding site on a HMW complex andNCoR) and which modulate the transcriptional repressive activityresulting from such interaction, may be identified.

[0052] Again, performance of an assay method according to the presentinvention may be followed by isolation and/or manufacture and/or use ofa compound, substance or molecule which tests positive for ability tointerfere with interaction between the HMW complex and NCoR and/orinhibit biological activity, i.e. transcriptional repressive activity.

[0053] An assay according to the present invention may also take theform of an in vivo assay. The in vivo assay may be performed in a cellline such as a yeast strain or mammalian cell line in which the relevantpolypeptides or peptides are expressed from one or more vectorsintroduced into the cell. In this case, the demonstration of theinteraction—or its prevention, or its disruption by the screenedcompounds—between the factors under study will have the form ofmeasurable enzymatic activity from a reporter enzyme, or fluorescence,or FRET phenoma.

[0054] Antibodies directed to the site of interaction in either the HMWcomplex or NCoR form a further class of putative inhibitor compounds.Candidate inhibitor antibodies may be characterised and their bindingregions determined to provide single chain antibodies and fragmentsthereof which are responsible for disrupting the interaction.

[0055] Antibodies generated during all of the previously describedassays may be obtained using techniques which are standard in the art.Methods of producing antibodies include immunising a mammal (e.g. mouse,rat, rabbit, horse, goat, sheep or monkey) with the protein or afragment thereof. Antibodies may be obtained from immunised animalsusing any of a variety of techniques known in the art, and screened,preferably using binding of antibody to antigen of interest. Forinstance, Western blotting techniques or immunoprecipitation may be used(Armitage et al, Nature 357:80-82, 1992). Isolation of antibodies and/orantibody-producing cells from an animal may be accompanied by a step ofsacrificing the animal.

[0056] As an alternative or supplement to immunising a mammal with apeptide, an antibody specific for a protein may be obtained from arecombinantly produced library of expressed immunoglobulin variabledomains, e.g. using lambda bacteriophage or filamentous bacteriophagewhich display functional immunoglobulin binding domains on theirsurfaces; for instance see WO92/01047. The library may be naive, that isconstructed from sequences obtained from an organism which has not beenimmunised with any of the proteins (or fragments), or may be oneconstructed using sequences obtained from an organism which has beenexposed to the antigen of interest.

[0057] Antibodies according to the present invention may be modified ina number of ways. Indeed the term “antibody” should be construed ascovering any binding substance having a binding domain with the requiredspecificity. Thus the invention covers antibody fragments, derivatives,functional equivalents and homologues of antibodies, including syntheticmolecules and molecules whose shape mimics that of an antibody enablingit to bind an antigen or epitope.

[0058] Example antibody fragments, capable of binding an antigen orother binding partner are the Fab fragment consisting of the VL, VH, C1and CH1 domains; the Fd fragment consisting of the VH and CH1 domains;the Fv fragment consisting of the VL and VH domains of a single arm ofan antibody; the dAb fragment which consists of a VH domain; isolatedCDR regions and F(ab′)2 fragments, a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

[0059] Following identification of a substance or agent which modulatesor affects the transcriptional repressive activity. e.g. the disruptionof the coiled coil region or similar oligomerization domains, thesubstance or agent may be investigated further. Derivatives with higheractivity, or improved pharmaco-kinetic properties, may be obtained;Furthermore, it may be manufactured and/or used in preparation, i.e.manufacture or formulation, of a composition such as a medicament,pharmaceutical composition or drug. These may be administered toindividuals.

[0060] Generally, a substance or agent which is capable of inhibiting,modulating or affecting the transcriptional repressive activity of theHMW complexes according to the present invention may be provided in anisolated and/or purified form, i.e. substantially pure. This may includebeing in a composition where it represents at least about 90% activeingredient, more preferably at least about 95%, more preferably at leastabout 98%. Such a composition may, however, include inert carriermaterials or other pharmaceutically and physiologically acceptableexcipients. As noted below, a composition according to the presentinvention may include in addition to an inhibitor compound as disclosed,one or more other molecules of therapeutic use, such as an anti-canceragent.

[0061] The present inventors have further determined that fusion of thecoiled coil region of PML to the human thyroid receptor (TR) results ina chimeric transcription factor with enhanced recruitment of NCoR, andenhanced transcriptional repressive properties (FIG. 8). It thereforefollows that oligomerization through the strong self-associating coiledcoil domain of molecules such as PML may enhance the functional activityof any given protein found in nature in a monomeric state, or in anunstable di- or multimeric state. The inventors have additionallyconsidered that the oligomerization of monomeric factors (or factors inan unstable di- or multimeric state) will result in the formation of anoligomeric complex with enhanced activity only in case of the properstructural organization of the oligomer itself, and of its activeinterfaces: for example, it must be assured (through the addition ofappropriate “hinge” regions) that the oligomerized interaction surfacesfor associated factors will be correctly and spatially oriented.

[0062] Thus, in a fourth aspect of the present invention, there isprovided a method of modifying the activity of a polypeptide bycontacting said polypeptide under suitable conditions with a compoundcomprising an oligomerization domain such as the coiled coil region ofPML. Preferably, the polypeptide is fused under suitable conditions withthe coiled coil region of PML or variants/derivatives thereof, with theintent of enhancing the biological activity of said polypeptide.

[0063] However, the present inventors have also determined that theoligomerization domain of p53 fused to RAR may substitute for the coiledcoil region of PML and as a result, shows enhanced recruitment of NCoR,enhanced transcriptional repressive properties and the block ofhematopoitetic differentiation. Thus, the present inventors havesurprisingly found that, although the nature of the oligomerizationdomain might differ from the coiled coil region of PML, the functionalproperties (for example, increased activity) remain the same.

[0064] By way of example, the present inventors have determined that thefollowing oligomerization domains, when fused to RAR, result in achimeric protein with increased activity and biological properties: a)the coiled coil region of PML; b) the oligomerization domain ofNPM-nucleophosmim; c) the POZ domain of PLZF; the oligomerization domainof NuMA; d) the tetramerization domain of p53.

[0065] The oligomerization domains present in the above mentionedproteins are also found (with variable degrees of homology) in otherproteins. For example, several proteins, including PML, are known whichshow the so-called tri-partite region, that includes a RING domain, aB-box(es) region, and the coiled coil (Saurin et al., 1996). Examples ofthese proteins, and of the corresponding coiled coil sequences are givenin FIG. 9.

[0066] The inventors believe that the coiled coil regions in proteinswhich correspond to PML, examples of which are given in FIG. 9, mediatea similar function to the coiled coil region in PML. Therefore, inaccordance with the present invention, the coiled coil regions of otherproteins (in addition to PML) may be fused to target polypeptides orproteins to enhance their functional activity.

[0067] Thus, the fourth aspect of the present invention may be expandedto include a compound comprising a coiled coil region of a protein, saidcoiled coil region corresponding to that of the coiled coil region ofPML. The protein may be one as exemplified in FIG. 9 or it may have,with regard to the coiled coil, structural and sequence similarity withthe coiled coil region of PML. Preferably, the sequence homology with beat least 50% homology in amino acid sequence with the coiled coil regionof PML. Preferably, the sequence homology will be at least 60%, morepreferably at least 70% and even more preferably at least 80% or 90%.

[0068] As mentioned above, the inventors have show that fusion of thecoiled coil domain of PML to RAR or TR increases the functional activityof these polypeptides. However, it will be appreciated by the skilledperson that the applicability of these coiled coil regions need not belimited to these particular polypeptides. Indeed, the coiled coilregions may be used to increase the functional activity of otherpolypeptides/ proteins such as: a) molecules endowed with enzymaticactivity, e.g. cre-recombinase, histone dacetylase; b) extracellularligands for cell membrane receptors; c) other transcription factors(nuclear receptors, HOX genes); and d) therapeutic antibodies. Thefunctional activity of such an antibody or part thereof (e.g. bindingdomain) will be increased owing to multiples of the antibody combiningto form a single compound.

[0069] The fusion of the coiled coil region to the polypeptide inquestion may be achieved using standard molecular techniques known tothe skilled person. For example, a plasmid may be generated to expressthe chimeric polypeptide/protein in bacteria or mammalian cells, wherethe coiled coil region or part thereof of, for example, PML is fused bystandard molecular biological techniques to the desiredpolypeptides/proteins. Upon fusion with the coiled coil region of, forexample PML, the chimeric protein may undergo in vitro and in vivoanalysis for their biochemical and functional properties. For example,the chimeric protein may undergo any one or more of the following:

[0070] 1) a demonstration of the oligomeric state of the chimericprotein compared with the natural protein;

[0071] 2) functional tests such as a) in the case of enzymaticactivities, enzymatic assays already available for the natural proteins;b) in the case of extracellular ligands, e.g. cytokines, the activationof the corresponding receptor, and the biological consequences of theactivation—e.g. apoptosis, differentiation, or other phenomena; c) inthe case of transcription factors, recruitment of coregulators (NCoR,HDACs, CBP/p300, P/CAF), and measurement in transfection assays oftranscriptional activity; d) in the case of therapeutic antibodies (orportions thereof), ELISA screening, neutralisation experiments orbiological assays.

[0072] Therefore, the present invention further provides a method ofenhancing the functional activity of a polypeptide, said methodcomprising producing a chimeric protein comprising a strongself-association domain (oligomerization domain) of a protein and saidpolypeptide, said chimeric protein not being present in nature as amultimeric complex. The strong self-association domain may be the coiledcoil domain of PML or it may comprise other domains such as exemplifiedin FIG. 9 which are related to the coiled coil domain of PML, or domainsconsisting of a different primary sequence and structure, but exerting asimilar function of induced oligomerization.

[0073] The present invention further extends in various aspects not onlyto a substance identified as a modulator of HMW complex formation andstability, or HMW complex/NCoR interaction or HMW complex/NCoR-mediatedactivity, property or pathway in accordance with what is disclosed.herein, but also a pharmaceutical composition, medicament, drug or othercomposition comprising such a substance, a method comprisingadministration of such a composition to a patient, e.g. for anti-cancersuch as leukaemia, use of such a substance in manufacture of acomposition for administration, e.g. for anti-leukaemia or similartreatment, and a method of making a pharmaceutical compositioncomprising admixing such a substance with a pharmaceutically acceptableexcipient, vehicle or carrier, and optionally other ingredients.

[0074] The invention further provides a method of modulating theactivity of HMW complexes which bind recruit and interact with NCoR, orother HMW complex mediated activity in a cell, which includesadministering an agent which inhibits or blocks the binding of HMWcomplex such as PML-RAR and AML 1-ETO to NCoR, such a method beinguseful in treatment of leukaemias or other diseases or disordersincluding malignancies where transcriptional repressive activity isimplicated.

[0075] The invention further provides a method of treating leukaemiaswhich includes administering to a patient an agent which interferes withthe binding of NCoR to HMW complexes comprising chimeric transcriptionfactors such as PML-RAR and AML 1-ETO.

[0076] The present inventors have further determined that the additionof the coiled coil domain of PML to a “target” protein may result infunctional inactivation of the target. They have shown that in the caseof proteins oligomeric in nature (such as wild type p53), addition of anextra oligomerization domain (the coiled coil of PML) results in anoligomerization chain reaction not compatible with normal p53localization and function. Thus, the inventors have determined thataddition of an extra-oligomerization interface (a coiled-coil inaccordance with the present invention) leads to the formation (throughthis oligomerization chain reaction) of high-order oligomeric.complexes, that results in the formation of non-functional aggregates.Put simply, the coiled-coil initiates a complexing of other oligomericfactors in its surrounding area which can serve to “mop up” (inactivate)unwanted protein in, e.g. a cell. The inventors have termed thistechnology “RITA” for Reaching (protein) Inactivation ThroughAggregation. The RITA technology may therefore be applied to inactivatenatural oligomeric proteins or any other protein that will be impairedfunctionally through this approach. Since the inventors have observedthat the CC domain may mediate oligomerization also in the extracellularenvironment, this technique may be applied to both intra- andextra-cellular target proteins.

[0077] Thus, in a fifth aspect of the present invention, there isprovided a method reducing the activity of a target oligomeric proteinin an environment comprising the step of introducing a modifiedoligomeric protein into the environment, said modified oligomericprotein being the same protein as the target, or a functional fragmentthereof, but comprising an additional oligomerization domain (coiledcoil domain).

[0078] The environment may be a sample comprising a population ofoligomeric proteins, e.g. an intracellular or extracellular environment.By addition of the modified oligomeric polypeptide to the sample, thoseoligomeric polypeptides present in the sample increase theiroligomerization state to undesired levels and as a consequence theiractivity within the sample is decreased. The newly derived modifiedoligomeric protein may not comprise the whole of the protein inquestion, as long as its natural oligomerization domain, and eventualother domains (for example, a nuclear localisation signal) required forputting in contact said modified oligomeric protein with its naturalcounterpart, are maintained. The environment may be intra-cellular orextracellular. If the environment is intra-cellular, the modifiedoligomeric protein may be introduced into the cell by transfecting thecell with a vector comprising nucleic acid encoding it and theoligomerization domain. For example, if the target protein was wild type(wt) p53, then it may be desirable to introduce a fusion proteincomprising the oligomerization domain and p53 (e.g. CC-p53). Such afusion protein, or nucleic acid encoding the fusion protein may form amedicament for use in reducing the activity or inactivating oligomericproteins in an environment e.g. a cell. Such medicaments may be used inthe treatment of patients or they may be used for research purposes. Forexample, the ability to “mop up” unwanted protein in a cell provides analternative to generating protein specific knock-out phenotypes. Thismay prove a faster and more practical preliminary test on the phenotypicimportance of a new gene. This is a far simpler and faster alternativeto generating knock out mice at the gene level, so often used now infunctional genomics studies. In addition, it has the potential to beapplied to human primary cells, or cell lines, and any other cellderived from a species for which the knock out technology is notavailable, or it is not ethically achievable (primates). Further, theyhave considerable application in the field of pharmacogenomics. Theinventors have concentrated their studies on p53. However, the personskilled in the art will appreciated that many oligomeric factors existand that therefore, this aspect of the invention may be applied to manyfactors e.g. cytokines, TNF, interleukins etc. The invention accordingto the fifth aspect may be used to “mop up” the wild type target proteinand inactivate it in large multimeric complexes. This application wouldbe particularly useful for inactivating systemic proteins involved inthe immune system e.g. TNF and interleukins. This sort of applicationmay be applied on a regulated and temporary basis to control graft ororgan transplantation rejection or to treat autoimmune disorders such asrheumatoid arthritis.

[0079] Thus, the present invention further provides an oligomerizationcomplex comprising an oligomeric factor and an oligomerization domain.The oligomeric factor is preferably selected from the group consistingof p53, interleukins, cytokines, TNF, etc. The oligomerization domain ispreferably the structural determinant for strong self-association andoligomerization of the oligomeric factors e.g. the coiled-coil domain.

[0080] The present invention further provides a nucleic acid molecule(DNA, cDNA; RNA, or mRNA) which encodes an oligomerization complex asdescribed above. The nucleic acid molecule may form part of anexpression vector and may be operably linked to a promoter which candirect the expression of the nucleic acid. Thus, the present inventionalso provides a replicable vector comprising sequence encoding anoligomerization complex. The invention further provides a host celltransformed with the vector described above.

[0081] Whether it is a polypeptide, antibody, peptide, nucleic acidmolecule (including vector/plasmid), small molecule, mimetic or otherpharmaceutically useful compound according to the present invention thatis to be given to an individual, administration is preferably in a“prophylactically effective amount” or a “therapeutically effectiveamount” (as the case may be, although prophylaxis may be consideredtherapy), this being sufficient to show benefit to the individual. Theactual amount administered, and rate and time-course of administration,will depend on the nature and severity of what is being treated.Prescription of treatment, e.g. decisions on dosage etc, is within theresponsibility of general practitioners and other medical doctors.

[0082] A composition may be administered alone or in combination withother treatments, either simultaneously or sequentially dependent uponthe condition to be treated.

[0083] Pharmaceutical compositions according to the present invention,and for use in accordance with the present invention, may include, inaddition to active ingredient, a pharmaceutically acceptable excipient,carrier, buffer, stabiliser or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material will depend on the route ofadministration, which may be oral, or by injection, e.g. cutaneous,subcutaneous or intravenous.

[0084] Pharmaceutical compositions for oral administration may be intablet, capsule, powder or liquid form. A tablet may include a solidcarrier such as gelatin or an adjuvant. Liquid pharmaceuticalcompositions generally include a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil.Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay be included.

[0085] For intravenous, cutaneous or subcutaneous injection, orinjection at the site of affliction, the active ingredient will be inthe form of a parenterally acceptable aqueous solution which ispyrogen-free and has suitable pH, isotonicity and stability. Those ofrelevant skill in the art are well able to prepare suitable solutionsusing, for example, isotonic vehicles such as Sodium Chloride Injection,Ringer's Injection, Lactated Ringer's Injection. Preservatives,stabilisers, buffers, antioxidants and/or other additives may beincluded, as required.

[0086] Examples of techniques and protocols mentioned above can be foundin Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed),1980.

[0087] Targeting therapies may be used to deliver the active agent morespecifically to certain types of cell, by the use of targeting systemssuch as antibody, cell specific ligands or viral vectors (in the case ofpolypeptides). Targeting may be desirable for a variety of reasons, forexample if the agent is unacceptably toxic, or if it would otherwiserequire too high a dosage, or if it would not otherwise be able to enterthe target cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

[0089]FIG. 1. Enhanced recruitment of NCoR by PML-RAR is due to thecoiled coil region of PML. Increasing amounts of GST -NCoR (from 150 ngto 10 μg) coupled to agarose beads were incubated with the indicated invitro translated, ³⁵-S labelled proteins. On the left, a schematicrepresentation of the in vitro translated products, with the tripartitemotif of PML highlighted (R, RING finger; B, B boxes; CC, coiled coil).The input lanes are loaded with the same amounts used in the pull-downexperiments.

[0090]FIG. 2. Role of the coiled coil of PML in the alteredtranscriptional properties of PML-RAR (A): Transcriptional repression byRAR and fusion proteins. Hela cells were co-transfected with theRARE-G5-TATA reporter in the absence (C) or presence of increasingamounts (50, 100, 250, 1000 ng) of the indicated expression vectors andthen harvested 48 hours after transfection. Levels of expression of RARand fusion proteins after transfection are shown in (C). (B) RARE andNCoR dependent transcriptional repression by RAR and fusion proteins.Hela cells were co-transfected with the RARE-G5-TATA or the G5-TATA,reporters and 1 μg of control expression vector (C), RAR (R), PML-RAR(P/R) and their respective AHT derivatives, as indicated. (D) RAsensitivity of RAR and fusion proteins. Cells were co-transfected with500 ng of the indicated expression vectors. 24 hours after transfection,RA was added at the following concentrations: 1, 10, 100 nM, 1, 10 μM,and cells were harvested after additional 24 hours.

[0091]FIG. 3. PML-RAR forms oligomeric complexes In vivo, which dependon the coiled coil of PML. (A) Role of the coiled coil of PML in theformation of HMW complexes. Nuclear extracts from NB4 cells or U937clones expressing the indicated proteins (P/R, PML-RAR; CC/R, CC-RAR;ΔCC/R, ΔCC-PML-RAR) were fractionated by gel filtration chromatography.Fractions were analysed by Western blotting using an anti-RAR antibody.Fraction number is indicated at the top of each lane: Elution fractionsof known molecular weight markers are indicated by arrows. (B)Recombinant PML-RAR forms oligomers. Fractions from gel filtration of invitro translated, ³⁵-S labelled (ivt), or highly purified, bacteriallyexpressed (BL21) PMl-RAR were analysed by SDS-PAGE, followed byautoradiography (ivt) or Western blotting using an anti-RAR antibody(BL21). (C) Biochemical purification of PML-RAR from U937 PR9 cells. Aschematic diagram of the purification scheme is presented, together withthe silver stain and the corresponding Western blot analysis of highlypurified PMl-RAR after DNA-affinity chromatography. The bands marked byan asterisk are non-specific bands present also in the control sample.The material eluted from the DNA affinity chromatography was alsoanalysed by gel filtration, followed by Western blot analysis. (D) Invivo cross-linking. Nuclear extracts from in vivo cross-linked,metabolically labelled U937 PR9 cells were analysed according to theflow-chart. Nuclear extracts were analysed by Western blot after SDSPAGE in reducing or non-reducing conditions: the arrows indicate thecross-linked species. Extracts were subjected to gel filtrationchromatography and then analysed by Western blot after SDS PAGE inreducing or not reducing conditions. The pool of HMW PML-RAR complexeswas immunoprecipitated with anti-PML or control (ctrl) antibodies andanalysed by autoradiography following SDS PAGE in reducing conditions.An aliquot of the anti-PML immunoprecipitate was analysed by gelfiltration, followed by SDS PAGE in reducing conditions andautoradiography. (E) Characterization of the oligomerization propertiesof the isolated coiled coil domain of PML. (Upper panel) Fractions fromgel filtration of purified, bacterially expressed (BL21) CC domain ofPML were analysed by SDS PAGE, followed by Western blotting using ananti-CC antibody. Purified CC was cross-linked in vitro with increasingconcentrations of BS³. The silver stain (lower left panel) and thecorresponding Western blot analysis (lower right panel) are shown. Theposition of the mono-, di- and tri-meric CC are indicated by arrowheads.

[0092]FIG. 4. PML-RAR oligomers associate with NCoR and DNA responsiveelements. (A) In vivo association of HMW PML-RAR complexes with NCoR.Samples from metabolically labelled U937 PR9 cells (I, input lane) wereimmunoprecipitated with anti-NCoR or control (PI) antibodies (note: thePI lane derives from approximately 5 times more material than thespecifically immunoprecipitated complexes). After extensive washing, theanti-NCoR containing beads were incubated in washing buffer in thepresence of RA (10 μM) for 2 hours at 4° C. (RA). For comparison, theimmunoprecipitate from the same cells using anti-PML antibodies is shownin the last lane (upper panel). The RA-eluted material was then analysedby gel filtration, followed by SDS PAGE and autoradiography (lowerpanel). (B) HMW PML-RAR complexes bind DNA. Mobility shift assays usingthe RARE from the RARβ2 promoter as a probe and extracts from Xenopusoocytes programmed with mRNA for RAR, PML-RAR either singly orco-injected with mRNA for RXR. Lanes 3-4, 5-8, 9-11 correspond to 0.03(lane 5), 0.05 (lanes 6, 9),0.2 (lanes 3, 7, 10) and 0.5 (lanes 4, 8,11) oocyte equivalent extract amounts. Arrows indicate the heterodimericRXR-RAR and PML-RAR/RXR complexes, and the HMW PML-RAR and PML-RAR/RXRcomplexes. (C) HMW PML-RAR complexes recruit NCoR on DNA. Mobility shiftassays on agarose gels using the RARE as a probe and extracts fromXenopus oocytes coinjected with mRNA for PML-RAR and RXR. Extracts wereincubated with the labeled RARE in the presence of recombinant GST-NCoR(aa 1782-2453) or GST as a control. Where indicated, RA (10 μM) wasadded during the incubation. (D) HMW PML-RAR complexes do not containNCoR or RXR. Western blot analysis using anti-NCoR (NCoR), anti-RAR(PML-RAR AHT) or anti-RXR antibodies (RXR) of fractions obtained fromgel filtration analysis of nuclear extracts from U937 cells (NCoR andRXR) or from PML-RAR AHT expressing U937 cells. An identical elutionprofile of NCoR and RXR was obtained from PML-RAR expressing cells (datanot shown).

[0093]FIG. 5. A heterologous oligomerization domain activates thetransforming potential of RAR. (A-B) p53-RAR form oligomers. (A) Nuclearextracts from COS-1 cells transfected with expression vectors forp53-RAR or RAR were incubated with tritiated RA (10 nM), fractionatedand analysed as described in FIG. 3A. Note that the low MW peak ofRA-binding capacity (coinciding with RAR) in p53-RAR cells derives fromexpression of wild type RAR, starting from its own ATG-conserved in thep53-RAR expression vector (see below)-. (B) Fractions from gelfiltration of in vitro translated, ³⁵-S labelled p53-RAR were analysedby SDS-PAGE, followed by autoradiography. Note that two products aregenerated in the reaction: full-length p53-RAR, fractionating inoligomeric complexes, and RAR, starting from the internal ATG site ofRAR and fractionating as a monomer. (C-D) Effects of RAR chimericproteins on differentiation of murine hematopoietic progenitors.Lin-cells were transduced with the indicated retroviral vectors and thensorted by FACS on the basis of their GFP positivity. In the case ofΔCC-PML-RAR, GFP+ cells were sorted in GFP^(high) or GFP^(low)expressors. After sorting, cells were either plated in differentiationmedium in the absence or in the presence of RA (3 nM or 1 μM) (C), oranalysed by Western blot (D). C, control, P-R, PML-RAR, ΔCC-P-R,ΔCC-PML-RAR, G-PR, GFP-PML-RAR, G-ΔCCPR, GFP-ΔCC-PML-RAR, G-p53R,GFP-p53-RAR. As described, RA delays myeloid differentiation of controlcells, leading to a maximum 20-30% reduction in the number of Mac1 +cells (Purton et al., 1999). This effect is counter-acted by expressionof PML-RAR and by high levels of ΔCC-PML-RAR. As described for wild-typeRAR, expression of high levels of ΔCC-PML-RAR in the presence ofphysiological concentrations of RA relieves the differentiation blockobserved in the absence of ligand (Du et al., 1999).

[0094]FIG. 6. APL fusion proteins form HMW complexes due to the partnersof RAR in the chromosomal translocations. (A) Nuclear extracts fromCOS-1 cells transfected with the indicated expression vectors were I)incubated with tritiated RA, fractionated and analysed as described inthe methods section or ii) fractionated without prior incubation withRA, and analysed by Western blotting using anti-RAR antibodies. (B)Fractions from gel filtration analysis of nuclear extracts from COS-1cells (NPM), or COS-1 cells transfected with PML or PLZF expressionvectors were analysed by SDS-PAGE, followed by Western blotting. (C) PMLis recruited to PML-RAR HMW complexes. Nuclear extracts from COS-1 cellsco-transfected with expression vectors for PML and PML-RAR werefractionated by gel filtration and then analysed by SDS-PAGE, followedby Western blotting with anti-PML (that do not cross-react with thefusion protein data not shown) or anti-RAR antibodies. Alternatively,extracts were co-immunoprecipitated with anti-RAR antibodies, and theimmunoprecipitated complexes were analysed by SDS-PAGE/Western, usinganti-PML antibodies (that recognize both PML and PML-RAR proteins,Flenghi et al., 1993).

[0095]FIG. 7. Oligomerization of AML 1-ETO. (A) A schematicrepresentation of AML 1-ETO and the deletions used: ZF, zinc fingers(NCoR interaction domain). (B) AML 1-ETO forms HMW complexes. AML1-ETOand ΔPC-AML1-ETO were in vitro translated, fractionated by gelfiltration chromatography (Superose 6, SMART system, Pharmacia Biotech)and analysed by SDS PAGE followed by autoradiography. (C-D) Interactionof AML1-ETO with NCoR and DNA. (C) AML 1-ETO and ΔPC-AML 1-ETO were invitro translated and then incubated with GST -NCoR (RDIII) or GST beads(C) as control in pull-down assays. Input lanes (I) represent 100% ofthe total. (D) extracts from AML 1-ETO U937 cells were incubated withbiotinylated oligos containing a specific AML 1 binding site or anunrelated sequence (Ctrl), and then pulled-down withstreptavidin-agarose beads (left panel). High-salt eluted material wassubjected to gel filtration chromatography, to verify that DNA-boundAML1-ETO was still present as HMW complexes (right panel). (E)Transcriptional repression by AML 1-ETO. C33A cells were transientlytransfected with the MDR1-luc reporter and the indicated expressionvectors. Luciferase activity was determined as described in the legendto FIG. 2. (F) Analysis of myeloid differentiation. Lin-cells transducedwith the indicated retroviral vectors were sorted and then treated asdescribed in the legend to FIG. 5.

[0096]FIG. 8. Fusion of the coiled coil of PML to a transcription factorleads to the formation of high molecular weight complexes, enhancedrecruitment of NCoR and enhanced transcriptional repression. (A) Thyroidreceptor (TR) or a chimeric thyroid receptor fused C-terminally to thecoiled coil of PML (CC-TR) were analyzed by gel filtrationchromatography after labeling of transiently transfected COS-1 cellswith iodinated thyroid hormone. (B) Increasing amounts of GST-NCoR (from150 ng to 10 μg) coupled to agarose beads were incubated with theindicated in vitro translated, ³⁵-S labelled proteins. On the left, aschematic representation of the in vitro translated products (CC, coiledcoil). (C) Transcriptional repression by TR and CC-TR. HeLa cells wereco-transfected with the TRE-G5-TATA reporter in the absence (control) orpresence of increasing amounts (50, 100, 250, 1000 ng) of the indicatedexpression vectors and then harvested 48 hours after transfection.

[0097]FIG. 9 shows example of sequences of coiled coil regions fromadditional proteins similar to PML in their primary sequence.

[0098]FIG. 10 shows the results of an experiment performed in U937 cellsexpressing PML-RAR under the control of an inducible promoter (Grignaniet al., 1998). The cells were either uninduced (ctr column), or inducedto express PML-RAR (PML-RAR column). Cells were transduced withretroviral vectors encoding the coiled coil of PML (RBCC) and GFP as amarker, or with retroviral vectors encoding GFP alone as a control(control). In control infections (gated in the cytofluorimetric analysisbased on the positivity for the GFP marker), induction of PML-RARstrongly inhibited differentiation by treatment of the cells withvitamin D and TGFβ (as assessed by percentage of cells expressing thedifferentiation marker CD14)—from >90% to about 30% of differentiated,CD14+ cells-. In cells expressing the coiled coil of PML (gated in thecytofluorimetric analysis based on the positivity for the GFP marker),PML-RAR was unable to block efficiently differentiation (from 90% to70%). Levels of expression of PML-RAR were unchanged by expressing thecoiled coil region of PML. Notably, the inventors did not noticeappreciable toxicity by expression of the coiled coil region of PML inthe uninduced cells.

[0099]FIG. 11 shows the result of investigations into the capacity ofcoiled coil-mediated oligomers to inactivate cellular proteins.

[0100] A: p53 (lane 1) and CC-p53 (lane 2) were in vitro translatedsingly, or co-translated (lane 3). Samples were then immunoprecipitatedwith antibodies against the coiled coil region of PML (lane 4, fromco-translated CC-p53 and p53; and lane 5, from p53 only), or with ananti-p53 specific antibody (lane 6, from p53 only).

[0101] B: Size exclusion chromatography (SEC) analysis of extracts from293 cells transiently transfected with expression vectors for p53,CC-p53, or both vectors. Whole-cell lysates were prepared and subjectedto SEC as described in the Materials and Methods section. Fractions wereanalysed by Western blotting using an anti-p53 antibody. Fraction numberis indicated at the top of each lane.

[0102] C: p53 null murine embryonic fibroblasts (MEFs) were transientlytransfected with a luciferase-based reporter vector for p53transcriptional activity. This vector contains multimerized p53 responseelements in front of a minimal promoter and of the reporter gene. Theexpression vectors indicated in the figure were co-transfected with thereporter and a β-galactosidase expression vector, used to normalize fortransfection efficiency. Twenty-four hours after transfection, cellswere collected and analyzed for reporter activity.

[0103] D: NIH 3T3 cells were transiently transfected with either anexpression vector for a GFP-p53 fusion protein (A: left panel, GFP-p53;right panel: DAPI staining), or for CC-p53 (B: left panel, staining withan anti-coiled coil antibody; right panel: DAPI staining). In panels Inparallel experiments, p53 was transfected in place of the GFP-p53chimeric protein, with identical results (data not shown). In panelsC-D-E, cells were co-transfected with CC-p53 and GFP-p53expressionvectors (C, CC-p53; D, GFP-p53; E, merge).

[0104] E: SAOS cells (p53 null) were transfected with either a controlvector (empty vector), or for vectors encoding p53, CC-p53, or both (1:1ratio). The vectors contain a G418 resistance marker. Forty-eight hoursafter transfection, cells were split, and plated in medium containingG418 to select for transfected cells. Ten-twelve days after plating,G418-resistant colonies were counted.

DETAILED DESCRIPTION

[0105] The Abnormal Recruitment of NCoR by PML. RAR is Caused by theCoiled Coil Region of PML

[0106] Recruitment of the NCoR-HDAC complex is crucial for thetransforming potential of PML-RAR. At low concentrations of RA (1-100nM), the stability of the PML-RAR-NCoR complex is higher than that ofthe RAR-NCoR complex (Grignani et al., 1998; He et al., 1998; Lin etal., 1998). Since PML does not interact directly with NCoR (Grignani etal., 1998), the present inventors investigated whether fusion to PMLaffected the stability of unliganded RAR for NCoR. Pull-down assays wereperformed by incubation of in vitro translated, ³⁵S labelled PML-RAR orRAR with GST-NCoR coupled to agarose beads. PML-RAR bound specificallyto the beads even at the lowest amounts of GST-NCoR tested, whereas atleast 15-30 fold higher amounts of GST-NCoR were required to obtainsignificant levels of RAR binding (FIG. 1). The inventors then mappedthe region(s) in the fusion protein responsible for the enhancedstability of the NCoR interaction: deletion of the PML coiled coilregion (ΔCC-PML-RAR) caused a dramatic decrease in the amount of boundprotein, giving a pattern of binding essentially identical to wild-typeRAR (FIG. 1). Deletion of other regions of PML (RING, B1 and B2 boxes)did not affect significantly the association of PML-RAR with NCoR (datanot shown). Conversely, fusion of the coiled coil region of PML to RAR(CC-RAR) resulted in a chimeric protein with the same characteristics ofbinding as PML-RAR (FIG. 1). These results show that PML-RAR binds NCoRwith higher apparent affinity than RAR and that the structuraldeterminant for this altered association is the coiled coil region ofPML.

[0107] The Coiled Coil Region of PML Determines the AlteredTranscriptional Properties of PML-RAR

[0108] The enhanced binding of PML-RAR to NcoR suggests that PML-RARmight act as a more potent transcriptional repressor than RAR. To testthis hypothesis, the inventors devised an artificial, reporter system tomeasure transcriptional repression by RAR and chimeric proteins. TheRARE-G5-TATA reporter construct has five GAL4 response elements fused toa minimal promoter region: upstream of the GAL4 sites, a RA responsiveelement (RARE) allows binding of RAR (or fusion proteins). Transienttransfection of this reporter in HeLa cells yielded low activity, whileco-transfection with an expression vector for the GAL4-VP16 activatorresulted in a strong response (10-13 fold induction; not shown). RARover-expression led to decrease of GAL4-VP16 activation, with 30-40%repression observed with the maximal amount of co-transfected RARexpression vector (1 μg, FIG. 2A), while it exerted no effect on theGAL4-VP16-mediated activation of a cognate promoter lacking the RARE(FIG. 2B). A RAR construct carrying the AHT mutation, which abrogatesNCoR binding (Horlein et al., 1995), was unable to prevent activation ofthe RARE-G5-TATA promoter by GAL4-VP16 (FIG. 2B).

[0109] Next, the inventors tested the transcriptional regulatoryfunctions of PML-RAR (or derivatives). Transfection of expressionvectors for RAR, PML-RAR or the various mutants yielded comparablelevels of protein expression (FIG. 2C). Whereas it was necessary totransfect at least 250 ng of the RAR expression vector to measuresignificant transcriptional repression, transfection of 50 ng of thePML-RAR expression vector resulted in relevant repression (30-40%). Atthe maximal concentration tested (1 μg), the inventors observed 80-90%repression with PML-RAR and 30-40% with RAR (FIG. 2A). Co-transfectionsof the PML-RAR expression vector with a RARE-less reporter abrogatedtranscriptional repression, and the PML-RAR AHT expression vector had noeffect on the activation of the RARE-G5-TATA promoter by GAL4-VP16,showing that—as for RAR—transcriptional repression by PML-RAR requiresDNA binding and is dependent on the recruitment of NCoR (FIG. 2B). TheΔCC-PML-RAR construct was identical to RAR in its capacity to weaklyrepress GAL4-VP16 driven transcription, whereas CC-RAR repressedGAL4-VP16 activity as strongly as PML-RAR (FIG. 2A).

[0110] The present inventors have previously shown that higherconcentrations of RA are required to dissociate PML-RAR (as opposed toRAR) from NCoR (Grignani et al., 1998). To address the role of thecoiled coil region on the stability of the association with NCoR in thepresence of RA, they performed the same transfection assays in thepresence of RA (1 nM-10 μM). Near-physiological RA concentrations (up to100 nM) reverted RAR and ΔCC-PML-RAR repression, whereas much higherconcentrations (1-10 μM) were required to revert repression by PML-RARand CC-RAR (FIG. 2C).

[0111] These results show that PML-RAR is a stronger transcriptionalrepressor than RAR, and its activity and altered RA sensitivity requirethe PML coiled coil region.

[0112] The Coiled Coil Region of PML is Responsible for the OligomericPML-RAR Complexes

[0113] Integrity of the coiled coil region is required for thebiological properties of PML-RAR (Grignani et al., 1996). The PML coiledcoil region is also responsible for the appearance of PML-RAR withinhigh molecular weight (HMW) complexes, as shown by gel filtrationanalysis of nuclear extracts from PML-RAR expressing cells (Grignani andal., 1999; Nervi et al., 1992). Fusion of the PML coiled coil region toRAR may change the composition of RAR-associated factors leading toenhanced recruitment of NCoR, transcriptional deregulation, andoncogenic activity. To investigate this possibility, the presentinventors analysed the molecular identity of these complexes.

[0114] The elution profile of PML-RAR was previously analysed byincubation of the extracts with tritiated RA and measurement of theradioactivity of RA-bound polypeptides (Benedetti et al., 1997; Nervi etal., 1992; Grignani et al., 1999). To eliminate the possibility that RAwould shift the identity of PML-RAR associated factors, the inventorsdecided to analyse unliganded complexes in fractions eluted from a gelfiltration column by Western blotting, using anti-RAR antibodies.

[0115] In extracts from all PML-RAR expressing cells examined (fresh APLblasts, and the promyelocytic NB4 cell line), unliganded PML-RAR waspresent in gel filtration fractions peaking with an apparent molecularweight of about 700 kDa (FIG. 3A and data not shown). This elutionvolume is identical to that previously observed using titrated RA (Nerviet al., 1992). In contrast, RAR always eluted as a monomeric species(FIG. 3A). Deletion of the PML coiled coil region shifted the elutionvolume of the PML-RAR complexes to regions corresponding to lowermolecular weight (mono- or dimeric) species, while CC-RAR was found inHMW complexes, confirming that the coiled coil domain of PML isnecessary and sufficient for the formation of HMW complexes (FIG. 3A).

[0116] To evaluate whether formation of HMW complexes is an intrinsicproperty of the fusion protein, the inventors analysed in vitrotranslated and bacterially expressed PML-RAR. They expressed andpurified PML-RAR in bacteria as an MBP-PML-RAR fusion protein and thenremoved the MBP moiety by factor Xa cleavage. Gel filtration analysisrevealed that in vitro translated and bacterially expressed PML-RAR wasstill found in HMW complexes (FIG. 3B), suggesting that the PML-RARnuclear complexes consist of oligomeric PML-RAR. To test thishypothesis, the inventors purified PML-RAR from nuclear extracts. Ourpurification scheme includes several chromatographic steps:heparin-Sepharose, Superose 6, and, as final step, DNA-affinity on abiotinylated RARE coupled to streptavidin-agarose beads. Only onespecific 120 kDa band (corresponding to PML-RAR, as shown by parallelWestern blot analysis) was observed after silver stain analysis of thepurified material (FIG. 3C). Gel filtration analysis revealed that theeluted PML-RAR was still present in HMW complexes (FIG. 3C), indicatingthat the HMW complex isolated from nuclear extracts reflect PML-RAR inoligomeric complexes.

[0117] To investigate whether the PML-RAR oligomeric complexes exist invivo, the inventors used the cell membrane-permeable, reversiblecross-linking agent DTBP in in vivo cross-linking experiments. Lysateswere prepared from in vivo cross-linked, metabolically labelled cells.SDS-PAGE analysis of the cross-linked material was performed undernon-reducing conditions (to preserve the cross-linking): in addition tothe 120 kDa PML-RAR band, a more intense, >350 kDa band, and a series ofless well resolved bands of higher MW (FIG. 3D), were recognised inWestern blot using anti-RAR antibodies. These last bands were absent ingels run in reducing conditions (to revert the cross-link) and from noncross-linked material (FIG. 3D and data not shown), and represented thecross-linked material. In parallel, the same nuclear extracts weresubjected to gel filtration: Western blot analysis from gels run inreducing or non-reducing conditions—of the fractions revealed that thecross-linked material was still present in HMW complexes (FIG. 3D). Thefractions corresponding to HMW complexes were then pooled andimmunoprecipitated using an anti-PML specific antibody. Theimmunoprecipitated complex contained exclusively one 120 kD ³⁵-Slabelled polypeptide (FIG. 3D), that was recognised by anti PML andanti-RAR antibodies (not shown), co-migrated with PML-RAR in SDS-PAGEand was absent in immunoprecipitates from samples prepared in identicalconditions from control cells (not shown). The inventors conclude thatthe labelled polypeptide represents PML-RAR, and that no other cellularproteins are stoichiometrically cross-linked under these conditions. Theinventors eluted the immunocomplexes by SDS 1% and then subjected theeluted PML-RAR to a new round of gel-filtration: interestingly, theyfound that PML-RAR was still present in HMW complexes (FIG. 3D). Thesame complexes were not recoverable from non cross-linked materialimmunoprecipitated from HMW complexes and then used as a control (datanot shown). Together, these results indicate that the oligomeric statusof PML-RAR pre-exist in vivo prior to cell lysis, and represents thenatural form of organisation of PML-RAR within the cell nucleus.

[0118] Estimation of the molecular mass of the PML-RAR oligomers by sizefractionation has intrinsic limitations. Indeed, the coiled coil regionof PML (although relatively small—approximately 100 residues—compared tothe total size of the protein—about 1,000 residues—) may influence theshape of PML-RAR (Hirano and Mitchison, 1994). By use of a complementaryapproach (centrifugation through a sucrose gradient), unliganded PML-RARsedimented at a position consistent with a MW 700 kDa (not shown).Calculation of the molecular mass of the oligomeric complex based onStokes radius (from gel filtration assays) and the sedimentationcoefficient (from sucrose gradients), the inventors could obtain anestimation of the molecular mass of PML-RAR oligomers withoutassumptions about the shape of the protein (Siegel and Monty, 1966):from those values, the inventors estimated a molecular mass of 650-700kDa, which is consistent with the formation of a PML-RAR hexamer.

[0119] There are no known cases, however, of coiled coil domainsmediating the formation of oligomeric hexamers (Lupas, 1996). To addressthis issue, we investigated the molecular properties of the isolatedcoiled coil domain of PML. The 14 kDa coiled coil domain (obtained bysite-specific cleavage of a purified GST-fusion protein) eluted as adefined HMW peak (60-150 kDa), confirming its capacity to oligomerize(FIG. 3E, upper panel). To assess its oligomerization number, wesubjected the isolated coiled coil to in vitro cross-linking studies.Treatment with two different cross-linkers (DT8P or BS³; see methods)resulted in the formation of higher MW bands, corresponding to di- andtri-meric species of the coiled coil domain (FIG. 3E and data notshown). Immunoblot experiments confirmed that the higher MW bandscorresponded to cross-linked coiled coil (FIG. 3E). Identical resultswere obtained by cross-linking experiments performed on the purifiedcoiled coil domain obtained from the gel filtration columns (data notshown). It appears, therefore, that the coiled coil domain can beisolated from bacteria as a trimeric complex, raising the question ofits relationship with the observed PML-RAR hexameric complex. Twopossible explanations can be envisaged: I) the oligomeric PML-RAR is atrimeric complex with different migration properties with respect to theglobular protein used as MW markers (Hirano and Mitchison, 1994; Lupas,1996); ii) the oligomeric PML-RAR complex is a trimer-trimer complex,due to additional protein-protein interactions mediated by other domainsof PML (RING, B-boxes) or RAR. In support of this latter hypothesis, wenoted that ΔCC-PML-RAR eluted as mono- and dimeric species (FIG. 3A),suggesting the presence of additional, coiled coil-independentinteractions among PML-RAR molecules. A two-step oligomerizationmechanism has been recently described for tenascin-C, where theformation of a parallel three-stranded coiled coil stabilizes theconnection of two triplets to a hexamer through an accessory interactiondomain (Kammerer et al., 1998).

[0120] PML-RAR Oligomers Recruit NCoR and RXR to RA—Responsive Elements

[0121] Recruitment of NCoR and RXR, as well as specific binding to DNA,are critical for the oncogenicity of PML-RAR (Minucci and Pelicci,1999). Therefore, the inventors investigated whether the oligomeric formof PML-RAR can associate with NCoR, RXR and specific DNA responsiveelements.

[0122] To investigate the association with NCoR, we analyzed anti-NCoRimmunoprecipitates from metabolically labeled nuclear extracts of cellsexpressing PML-RAR. An approximately 120 kDa protein co-precipitatedwith NCoR (FIG. 4A). We identify the 120 kDA, NCoR-associated factor asPML-RAR, based on the following criteria: I) it co-migrated with PML-RAR(as determined by parallel immunoprecipitation of the same nuclearextracts with anti-PML or anti-RAR antibodies: FIG. 4A and data notshown); ii) it was not present in the immunoprecipitates from cells notexpressing PML-RAR (data not shown); and iii) it could be specificallydissociated from the NCoR immunoprecipitated complexes by incubationwith RA (FIG. 4A). To determine whether the oligomeric form of PML-RARwas able to associate with NCoR in vivo, we performed gel filtrationanalysis of the RA-eluted fraction from anti-NCoR immunoprecipitates. Asshown in FIG. 4A, lower panel, PML-RAR was recovered in HMW complexes,demonstrating the existence of an oligomeric PML-RAR/NCoR complex invivo.

[0123] To establish whether PML-RAR oligomers bind DNA specifically, theinventors expressed PML-RAR (or RAR) into Xenopus oocytes. This systemhas been widely used to study the transcriptional regulatory functionsof nuclear receptors (including RARs: Wong et al., 1998; Minucci et al.,1998) and contains low to undetectable levels of endogenous receptors(unlike mammal cells), thus allowing unambiguous evaluation of the DNAbinding properties of exogenous receptors (Wong et al., 1996; Minucci etal., 1998). Since RAR and PML-RAR have been previously shown to requireRXR for high efficiency binding to DNA responsive elements, in someexperiments we coinjected mRNAs for RXR to express RXR/RAR orPML-RAR/RXR complexes. Extracts from injected oocytes were tested inmobility shift assays, using the RA responsive element (RARE) from theRARβB2 promoter as a probe. As previously shown, injection of mRNA forRAR resulted in no detectable binding complex, whereas coinjection ofmRNA for RXR caused the formation of a strong heterodimeric RXR/RAR DNAbinding complex (FIG. 4B; Minucci et al., 1998). Expression of PML-RARresulted in the formation of a complex (complex I) which migratedconsiderably more slowly than the heterodimeric RXR-RAR complex (FIG.4B). This complex was specific, since it was competed by an excess ofcold RARE (but not by an unrelated oligonucleotide: data not shown). Toestablish conclusively whether the PML-RAR/DNA slowly migrating complexcorresponds to HMW PML-RAR bound to DNA, the present inventors performedsize exclusion chromatography analysis of the radioactively labelledRARE/PML-RAR complex in the same buffer conditions as the mobility shiftassays. In the absence of cell extracts, or after incubation withcontrol extracts lacking PML-RAR, the RARE was completely absent infractions corresponding to a predicted MW of 30-60 kDa, consistent withits length and size (37 double-stranded oligonucleotide). In PML-RARcontaining extracts, however, a new peak of radioactive RARE wasobserved, precisely co-fractionating with PML-RAR and corresponding toan apparent MW>670 kDa (data not shown). Parallel analysis of the samesamples in mobility shift assays revealed the appearance of thePML-RAR/DNA slowly migrating complex, showing that this complex indeedrepresented the binding of HMW PML-RAR to the DNA. As for RAR,coexpression of RXR resulted into enhanced binding to DNA (FIG. 4B).Notably, two novel complexes were formed in this case: I) a lowabundance complex (complex II), that migrated slightly slower than theRXR/RAR heterodimer and that we interpret as the heterodimer formed by(monomeric) PML-RAR and RXR; ii) a highly abundant complex (complexIII), that migrated much more slowly than complex II and slightly slowerthan complex I and that we interpret as the oligomeric PML-RAR/RXR DNAbinding complex (FIG. 4B).

[0124] Further mobility shift assays were performed to determine whetherNCoR might be recruited to the PML-RAR/RXR/DNA complex. We used agaroseas a solid matrix for the electrophoretic runs in this case, to allowbetter resolution of very high molecular weight complexes (in the rangeof 500 kDa-1 MDa). The oligomeric PML-RAR/RXR/DNA complex wassuper-shifted by the addition of recombinant GST-NCoR(but not controlGST: FIG. 4C). RA addition caused the disappearance of the supershiftand the formation of an oligomeric PML-RAR/RXR/DNA complex that migratedslightly faster than the complex observed in the absence of RA (FIG. 4C,lane 2 against lane 5). It has been previously observed that ligandbinding to RAR and other nuclear receptors results in a faster migrationof the receptor/DNA binding complex, likely as a result of aconformational change in the receptor ligand binding domain induced byRA (reviewed in Chambon, 1996). These results demonstrate that PML-RARoligomers bind DNA, that DNA binding is enhanced by RXR recruitment, andthat NCoR may be recruited to the oligomeric PML-RAR/RXR/DNA complex.The association of PML-RAR oligomers with NCoR and RXR does notcontradict the inventors' finding that PML-RAR HMW complexes originateexclusively from oligomerization of the fusion protein, in the absenceof other cellular proteins interacting stoichiometrically (FIG. 3). Infact, neither the NCoR/HDAC complex nor RXR co-fractionated with PML-RARin gel filtration of lysates from PML-RAR expressing cells, and thePML-RAR AHT mutant, that is unable to recruit the NCoR/HDAC complex, hasan elution profile coinciding with PML-RAR (FIG. 4D). It appears,therefore, that PML-RAR can be isolated as tightly interacting,self-associating oligomeric complexes which represent the “core” complexresponsible for the interactions (at lower affinity and/orstoichiometry) with other factors, such as nuclear corepressors and RXR.

[0125] Fusion with a Heterologous Oligomerization Domain Increases NCoRBinding and the Transcriptional Repressive Activity of RAR and Activatesits Leukaemogenetic Potential

[0126] The inventors' results point to a critical role for the coiledcoil region of PML in mediating oligomerization, which represents thestructural determinant for the aberrant interaction with the NCoR/HDACcomplex and for leukemogenetic activity of the fusion protein. Todetermine whether the oligomerization per se is the critical function,or if other properties of the PML coiled coil region play a role, theinventors evaluated the effects of a heterologous oligomerization domainon the transcriptional and biological properties of RAR. To this end,they fused RAR C-terminally to the tetramerization domain present inp53, a well-studied self-association module that allows tetramerizationof heterologous proteins(Chen et al., 1998; Clore et al., 1994). COS-1cells were transfected with a p53-RAR expression vector, and nuclearextracts were labelled with tritiated RA. RA binding capacity wasanalysed by gel filtration chromatography: the profile obtained showedthat p53-RAR formed HMW complexes (FIG. 5A). The fusion protein was thenin vitro translated and analysed by gel filtration chromatography:compared to RAR, which elutes as a monomer, p53-RAR was found in HMWcomplexes (FIG. 5B). These results confirm that the oligomerizationdomain of p53 is able to impose the self-association of RAR, allowing usto evaluate the biological activity of RAR oligomers that do not containPML sequences..

[0127] The inventors performed pull-down assays by incubation of invitro translated, ³⁵S labeled p53-RAR with increasing concentrations ofGST-NCoR. Similarly to PML-RAR, p53-RAR bound NCoR even at the lowestamounts of GST-NCoR tested (FIG. 1). The inventors then measured thecapacity of p53-RAR to repress GAL4-VP16 driven transcription: as shownin FIG. 2A, p53-RAR repressed GAL4-VP16 activity as strongly as PML-RARand the CC-RAR mutant, and was a more potent transcriptional repressorthan the natural RAR.

[0128] The capacity of p53-RAR to block differentiation was evaluatedusing primary hematopoietic precursors purified from murine bone marrowon the basis of the absence of surface differentiation antigens (lin-seemethods). Lin-cells were transduced using retroviral constructs encodingfor PML-RAR (or derivatives) and GFP as a marker: Cells transduced withthe control retroviral vector-expressing GFP only—behaved identically touninfected cells (data not shown). GFP-positive cells were sorted andseeded in methylcellulose plates containing a cytokine cocktail(including G-CSF and GM-CSF), to allow terminal myeloid differentiation.After 8-10 days, colonies were pooled and analysed for the expression ofmyeloid differentiation markers (Mac-1 and GR-1). The results for Mac-1are presented in FIG. 5C: similar results were obtained for GR-1 (datanot shown). Compared with control cells, PML-RAR expressing cells showeda strong reduction in their capacity to differentiate (FIG. 5C). Cellsexpressing ΔCC-PML-RAR showed—upon sorting—much higher levels ofexpressed protein compared to PML-RAR, as judged by Western blot (datanot shown and FIG. 5D). It has been shown that high levels of RAR leadto a differentiation block. This is likely to be owing to sequesteringof available RXR (Du et al., 1999; Grignani et al., 1996). For thisreason, the inventors sorted the ΔCC-PML-RAR GFP+ cells into twopopulations, according to their mean fluorescence levels:correspondingly, Western blot analysis revealed that GFP^(low) cellsexpressed lower levels of ΔCC-PML-RAR than the GFP^(high) cells,although the level of the chimeric protein was at least 2-3 fold higherthan PML-RAR (FIG. 5D). ΔCC-PML-RAR had essentially no effects ondifferentiation of the GFP^(low) infected cells, whereas at higherlevels it induced a consistent differentiation block (>30%, compared toabout 50% block for PML-RAR). Lin-cells were then infected with aretroviral construct encoding p53-RAR as a GFP-fusion protein. Infectedcells were sorted and plated in methylcellulose differentiation mediumas described before. The inventors observed a strong decrease in thenumber of Mac1+ and GR1+ cells in the p53-RAR infected sample comparedto control cells, similarly to what was observed upon PML-RAR orGFP-PML-RAR expression (FIG. 5C and data not shown). At comparablelevels of expression, a GFP-ΔCC-PML-RAR construct had no effects on thedifferentiation of transduced cells (data not shown). Taken together,these observations demonstrate that addition of an oligomerizationdomain to RAR is sufficient to obtain a fusion protein with fulltransforming potential.

[0129] The present inventors then compared the capacity of PML-RAR,CC-RAR, and p53-RAR to mount a RA-response in transduced murine primaryhaemopoietic precursors, measuring the capacity of RA to relieve thedifferentiation block due to expression of RAR-fusion proteins. Thedifferentiation block by PML-RAR and CC-RAR was relieved exclusively athigh concentrations of RA (1 μM, FIG. 5C). In contrast, p53-RARexpressing cells were insensitive to RA treatment at all concentrations(FIG. 5C). Similar results were obtained in p53-RAR U937 cells,excluding the contribution of cell-type specific effects (data notshown). Accordingly, RA did not relieve transcriptional repression byp53RAR on a RARE-containing reporter, in contrast with PML-RAR andCC-RAR that were fully responsive at high RA concentrations (FIG. 2D).Use of other RAR ligands (9-cis-RA, TTNPB) resulted in identical results(data not shown). Scatchard analysis performed to measure the affinityof RA for p53-RAR yielded a calculated apparent equilibrium dissociationconstant (Kd) of 4.0+/−0.42 nM (mean +/−SD, n=3), compared to 0.5 nMobserved for RAR, PML-RAR and CC-RAR in parallel samples (data notshown). Thus, the affinity of RA for p53-RAR is lower than for PML-RAR.At the concentrations of RA used in the experiments above (1-10 μM;4,000 higher than the measured Kd), however, this difference is unlikelyto be significant. It appears, therefore, that RAR-fusion proteinoligomers exert differential responses to RA on the basis of theidentity of the oligomerization domain fused to RAR.

[0130] PML, PLZF and NPM Form HMW Complexes In Vivo and InduceOligomerization of their Corresponding RAR Fusion Proteins

[0131] Other RAR fusion proteins (such as PLZF-RAR and NPM-RAR) areinfrequently associated with the promyelocytic leukemia phenotype(Melnick and Licht, 1999). They share with PML-RAR the same portion ofRAR and the ability to block differentiation, to recruit the NCoR-HDACcomplex and to deregulate expression from RA-target genes (Minucci andPelicci, 1999; Redner et al., 1999). Although analysis of PLZF and NPMsequences failed to identify putative coiled coils, both PLZF and NPMcontain strong protein-protein interaction domains directing theirself-association and retained within the corresponding fusion proteins(Ahmad et al., 1998; Chan and Chan, 1995). PLZF-RAR has been shown toform HMW nuclear complexes (Benedetti et al., 1997). The presentinventors investigated whether the three RAR translocation partners PML,PLZF and NPM are able to form HMW nuclear complexes.

[0132] PML is tightly associated to the nuclear matrix, and was notextracted in the inventors' experimental conditions (Chang et al.,1995). Over expression of PML provokes a partial solubilization, andapproximately 10-20% of the protein is then found in the nucleoplasmicfraction of nuclear extracts (data not shown). Gel filtration analysisof nuclear extracts from transfected HeLa cells revealed that PML isdistributed in HMW complexes with an apparent MW ranging from >670 kDato the void volume of the column (FIG. 6B). Identical results wereobtained by expressing PML in bacteria, indicating that the HMWcomplexes correspond to oligomeric PML forms (data not shown). PLZF andNPM were also found in HMW complexes (FIG. 6B). PLZF peaked at anapparent MW>440 kDa, whereas NPM was found in two distinct pools, as amonomer (30% of total NPM) or in HMW complexes (ranging approximatelyfrom 200 to >400 kDa) consistent with an hexameric state, as previouslydescribed (Chan and Chan, 1995). Gel filtration analysis ofNPM-RAR-expressing nuclear extracts labeled with tritiated RA showeddistribution patterns similar to those of PML-RAR and PLZF-RAR, althoughit peaked with a slightly lower apparent MW (400 kDa), consistent withthe lower MW of NPM-RAR compared to PML-RAR (60 kDa versus 120 kDa, FIG.6A). It appears, therefore, that RAR-fusion protein all form HMWcomplexes in vivo through their corresponding PML, PLZF or NPM moieties.

[0133] The ability of the PML coiled coil region (unlike theoligomerization domain of p53) to restore the RA response suggests thatit may recruit additional nuclear factors to PML-RAR oligomers. Theinventors therefore looked at the possibility that PML itself might berecruited to PML-RAR complexes. Gel filtration chromatography ofextracts from HeLa cells transiently transfected with PML and PML-RARexpression vectors showed co-fractionation of PML and PML-RAR (FIG. 6C).Analysis of anti-RAR immunoprecipitates from fractions containing PMLand PML-RAR revealed the presence of PML, showing that PML can berecruited to PML-RAR HMW complexes (FIG. 6C). RA treatment did notmodify PML-RAR complexes or affected PML recruitment to PML-RARoligomers (data not shown): therefore, PML is a candidate PML-RARco-factor in the RA-response of APL cells.

[0134] Oligomerization of the AML1/ETO Fusion Protein

[0135] Mutation of the NCoR binding site impairs the biological activityof AML 1-ETO (Gelmetti et al., 1998). In this case, recruitment ofNCoR-HDAC is mediated by ETO and might be sufficient to alter thefunction of AML 1. However, an AML1-HDAC1 fusion protein was unable toblock hematopoietic differentiation (not shown), suggesting thatrecruitment of HDAC is not sufficient to activate the oncogenicpotential of AML 1. Analysis of the ETO primary sequence revealed twoputative protein-protein interaction domains: a coiled coil region (PC1,residues 444-492) and an amphipatic α-helix (PC2, residues 352-378;Lutterbach et al., 1998) (FIG. 7A). Since ETO has been shown previouslyto form HMW complexes (Lutterbach et al., 1998), the inventors performedgel filtration analysis of AML 1-ETO and of a defective mutant of thefusion protein lacking PC1-PC2 (ΔPC-AML1-ETO in FIG. 7A). AML 1-ETO wasfound within HMW fractions, while deletion of PC1 and PC2 regionsshifted the fusion protein to lower molecular weight forms (FIG. 7B anddata not shown). AML1a was found as monomeric species, confirming therequirement for ETO in the formation of HMW complexes by the fusionprotein (data not shown). As a further characterisation of the HMWcomplexes, the inventors expressed and purified AML 1-ETO from bacteriaas an MBP-AML 1-ETO fusion protein and then removed the MBP moiety byfactor Xa cleavage. Gel filtration analysis revealed that bacteriallyexpressed AML 1-ETO formed HMW complexes identically as the in vitrotranslated form, indicating its oligomeric state (data not shown). Theinventors then investigated if the loss of the capacity to form HMWcomplexes correlated also with changes in the ability of AML 1-ETO torecruit NCoR and to repress transcription. The ETO interaction site forNCoR has been mapped in vitro at the two C-terminal zinc finger motifs(Gelmetti et al., 1998; Lutterbach et al., 1998). Deletion of the PC1and PC2 motifs led to a strong decrease in the amount of fusion proteinbound to GST-NCoR, despite the fact that the NcoR binding site isretained in the APC-AML1-ETO fusion protein (FIG. 7C). Since the PCmotifs and NcoR do not interact in vitro (unpublished results), thesedata indicate that the PC motifs contributes to NCoR recruitment byAML1-ETO through their ability to mediate the formation of HMWcomplexes.

[0136] AML 1-ETO has been shown to bind DNA alone or as AML 1-ETO/CBFβcomplexes (Meyers et al., 1995). Since the DNA binding complex is theeffector of the leukemogenic effect of AML 1-ETO, the inventors analyzedwhether HMW AML 1-ETO complexes are able to bind DNA. To this end, wepartially purified AML 1-ETO from AML 1-ETO expressing cells by DNAaffinity (using a specific AML 1 specific response element; see methodsand FIG. 7D, left panel). Gel filtration of DNA-eluted material showedthat AML 1-ETO can be recovered in its oligomeric form after DNA binding(FIG. 7D, right panel), suggesting that the oligomeric AML 1-ETO/DNAcomplex might recruit NcoR and efficiently repress transcription.Consistently, deletions of either the NCoR binding site (ΔZF-AML1-ETO)or the oligomerization regions (ΔPC-AML1-ETO) greatly impaired thecapacity of the fusion protein to: I) repress transcription from atarget promoter—MDR-1—in transient transfection assays (FIG. 7E andLutterbach et al. , 1998); and ii) block differentiation of primaryhemopoietic progenitors (FIG. 7F). Taken together, these resultsindicate that the efficient recruitment of the NCoR-HDAC complex byAML1-ETO is required to activate the oncogenic potential of AML1, andthat this is achieved by the formation of AML1-ETO HMW complexes.

[0137] Fusion of the Coiled Coil Domain of PML to a HeterologousTranscription Factor Enhances its Functional Activity

[0138] The inventors' findings point to a critical role for the fusionof the coiled coil domain of PML in altering the functional activity andinducing the oncogenic potential of RAR. To investigate ifoligomerization would likewise enhance/alter the function of otherfactors, they generated a chimeric protein where the coiled coil domainof PML was fused to the entire coding sequence of the human thyroidreceptor (TR). TR belongs to the superfamily of nuclear hormonereceptors, and—as RAR—repress transcription by recruiting NCoR/HDAC inthe absence of ligand. CC-TR was found in HMW complexes after gelfiltration of in vitro translated reaction products, whereas TR elutedas a predominant monomeric fraction (FIG. 8A). This result showsthat—upon fusion—the coiled coil region of PML is able to induce theformation of HMW complexes of heterologous factors. Interestingly, CC-TRshowed a much stronger interaction with NCoR (FIG. 8B) and an enhancedcapacity to repress transcription (FIG. 8C) compared to TR, indicatingthat oligomerization through the coiled coil enhanced its functionalactivity. Thus, oligomerization of a factor through fusion with thecoiled coil of PML appears to be a (potentially) generally availablemethod to enhance its function. Since coiled coil-medicated oligomerscan form also in the extra-cellular environment (data not shown), thisapproach may be applicable to both intra- and extra-cellular peptides.

[0139] Disruption of PML-RAR Oligomers Prevents Differentiation Block

[0140] RAR oligomerization through the coiled coil region of PML isrequired for its leukemogenic activity, and deletion of the PML coiledcoil is sufficient to lead to loss of oncogenic potential (Minucci etal., 2000). Small molecule compounds able to disrupt PML-RAR oligomerswould therefore be able to revert the leukemic phenotype. As a proof ofprinciple, we demonstrated here that over-expression of the coiled coilof PML (including the additional amino-terminal region required fortargeting to the appropriate nuclear compartment)—by associating withPML-RAR and therefore reducing its oligomerization—achieved ananti-leukemic effect (FIG. 10).

[0141] The Capacity of Coiled Coil-Mediated Oligomers to InactivateCellular Proteins

[0142] Fusion of the PML coiled coil to a heterologous factor results ina chimeric protein with altered properties. In the case of PML-RAR andCC-RAR, the net outcome is a transcription factor with enhanced capacityto recruit co-regulators. So, it would appear that oligomerization hasthe capacity to enhance the biochemical properties of a given natural(or artificial) monomeric factor. Several factors (such as PML itself)are oligomeric in nature. In this case, addition of anextra-oligomerization interface would lead to formation (through anoligomerization chain reaction) of high-order oligomeric complexes, thatmay result in the formation of non-functional aggregates. The inventorsinvestigated whether this may constitute a generally applicable approachfor the functional inactivation of a given “target” protein, and termedthis technology “RITA” (for “Reaching (protein) Inactivation ThroughAggregation”). The oncosuppressor p53 protein forms tetramers, andoligomerization is required for its function. In the CC-p53 chimericprotein, the oligomerization domain of PML (CC) fused to the full-lengthcoding sequence of p53 should impose an altered oligomerization statenot only of the chimeric protein, but also of wild-type, interactingp53. In turn, this—according to the inventors' model—should lead to animproper organization of CC-p53/wt p53 hetero-oligomers, and toinhibition of p53 function.

[0143] The inventors fused the CC region of PML to the full-length p53,to generate the chimeric CC-p53 protein. A necessary requirement for thechimeric CC-p53 protein would be the capacity to interact with wt p53,through the p53 tetramerization domain present in both proteins.Antibodies directed against the CC region of CC-p53 were able toimmunoprecipitate in vitro translated p53 only in the presence of theCC-p53 chimera, showing the existence of a CC-p53/wt p53 complex (FIG.11A).

[0144] P53 forms stable tetramers, as observed after size exclusionchromatography (SEC). Consistently with the presence of an additionaloligomerization interface, CC-p53 is found in SEC fractions of muchhigher apparent molecular weight, of approximately 600 kDa (FIG. 11B).Given the capacity of CC-p53 to associate also with wt p53, theinventors measured the apparent molecular weight of the CC-p53/p53hetero-oligomeric complex by SEC. Upon interaction with CC-p53, p53 wasfound to co-fractionate with the chimeric protein, (FIG. 11B).Co-immunoprecipitation experiments performed on the pooled fractionscorresponding to the peaks of CC-p53 and wt p53 showed the formation ofa hetero-oligomeric CC-p53/p53 complex, demonstrating that CC-p53 isable to recruit p53 into high-order oligomers (data not shown). Theinventors did not notice a change in the elution profile of the CC-p53chimera upon co-expression of the p53, suggesting that thehetero-oligomeric CC-p53/p53 complexes do not differ significantly fromCC-p53 oligomers in size. To evaluate the transcriptional properties ofCC-p53, the inventors performed transient transfection assays in murineembryonic fibroblasts (MEFs) derived from p53-/-mice. In these cells,transfection of a p53 reporter construct resulted in minimal levels oftranscriptional activity (FIG. 11C). Co-transfection of an expressionvector for wt p53 caused a strong increase in transcriptional activityof the reporter construct (50-100 fold: FIG. 11C). Co-transfection of anexpression vector for CC-p53, in contrast, had no effect on reporteractivity, showing that the chimeric protein is no longer able toregulate p53 target genes (FIG. 11C). Interestingly, co-transfection ofincreasing amounts of CC-p53 with wt p53 (at a fixed amount) resulted ina dramatic repression of wt p53 transcriptional activity (FIG. 11C). Asa control, the chimeric CC-VDR protein, encoding for an unrelatedtranscription factor (vitamin D receptor) fused to the PML coiled coil,had little or no effect on p53 wt transcriptional activity (FIG. 11C).It appears therefore that CC-p53, interacting with wt p53, is able toblock its transcriptional activity. Next, the investigated more indetails the mechanism(s) underlying the dominant negative effect ofCC-p53 over the wt p53 protein. The wt p53 protein ispost-translationally regulated at several levels: stability,phosphorylation, acetylation.

[0145] The inventors first asked whether the hetero-oligomeric CC-p53/wtp53 complexes are less stable than the wt p53 protein: Western blotanalysis of cells transiently transfected with the expression vector forwt p53, or co-transfected with the expression vectors for wt p53 andCC-p53, showed no significant difference in wt p53 levels, suggestingthat CC-p53 is not targeting wt p53 for degradation(in conditions wherep53 transcriptional activity is strongly repressed: data not shown).Next, The inventors checked for proper localization of thehetero-oligomeric complexes. NIH 3T3 cells were transiently transfectedwith expression vectors for wt p53, CC-p53, and—in some experiments—aGFP-p53 fusion protein, to allow visualization of p53 prior fixation ofthe cells, and to distinguish unambiguosly p53 from CC-p53. GFP-p53behaves identically to wt p53 in all functional assays tested (data notshown). GFP-p53 and p53 displayed a typical, nuclear localizationpattern (FIG. 11D, panel A, and data not shown). In contrast, CC-p53 wasalmost entirely localized in the cytoplasm (FIG. 11D, panel B).Strikingly, CC-p53 caused complete delocalization of either wt p53, orGFP-p53 (FIG. 11D, panels C-E). These results suggest that the dominantnegative effect of CC-p53 over. wt p53 is mainly achieved throughformation of hetero-oligomeric complexes unable to enter the nucleus.

[0146] Finally, the inventors measured the capacity of CC-p53 to inhibitthe biological function of p53. Expression of wt p53 in p53 null SAOScells results in cell growth arrest, apoptosis, and loss of colonyforming capacity (FIG. 11E). CC-p53 had no effect on cell viability(FIG. 11E). Strikingly, CC-p53 almost completely abrogated the growthsuppression effect by wt p53, demonstrating that the dominant negativeeffect on wt p53 activity is sufficient to inhibit its biologicalfunction (FIG. 11E).

[0147] Taken together, these results show that addition of the coiledcoil domain of PML to a “target” protein results in functionalinactivation of the target. The inventors have shown that in the case ofproteins oligomeric in nature (such as wt p53), addition of an extraoligomerization domain (the coiled coil of PML) results in anoligomerization chain reaction not compatible with normal p53localization and function. The RITA technology may therefore be appliedto inactivate natural oligomeric proteins. Since the inventors haveobserved that the CC domain may mediate oligomerization also in theextracellular environment, this technique may be applied to both intra-and extra-cellular target proteins.

[0148] Discussion

[0149] The main results presented here are that PML-RAR forms nuclearoligomers in vivo, and that oligomerization of RAR (through fusion withthe PML coiled coil region or with the p53 oligomerization domain) leadsto deregulated transcription from RA-target promoters anddifferentiation block when expressed into primary hematopoieticprogenitor cells. The present inventors propose that oligomerization isthe mechanism responsible for the oncogenic activation of RAR uponfusion with PML.

[0150] As effectors of the RA signal, natural RARs directly regulate theexpression of a variety of target genes, both in the absence (asrepressors) and in the presence (as activators) of ligand (Minucci andPelicci, 1999). Transcription from RA-target genes is an even morecomplex phenomenon, since several other intracellular signaling pathwaysand transcription factors contribute to their regulation (Mangelsdorfand Evans, 1995; Minucci and Ozato, 1996). Therefore, transcription fromRA-target genes represents, at any given time-point and for each targetpromoter, the result of a “concerted” mode of transcriptionalregulation, resulting from the cooperation among different DNA-bindingproteins and associated co-regulators (Kadonga, 1998; Ptashne and Gann,1997; Tjian and Maniatis, 1994). The inventors' findings show thatoligomerization is sufficient to subvert this regulatory network bymarkedly enhancing the capacity of a transcription factor to recruitco-regulators, and leads to an “a solo” mode of deregulatedtranscription. The inventors hypothesize that oligomerization of RARresults in the recruitment of over-physiological concentrations oftranscriptional corepressors, leading to a chromatin configuration whichmay render the target promoter's refractory to activating signals fromother cis-regulatory elements (constitutive transcriptional repression).This model represents the explanation at the molecular level of theoncogenic activation of RAR in APL, and a framework for the futureanalysis of expression patterns of target genes deregulated by thefusion protein.

[0151] In several cases, transcription factors have been shown tooligomerize physiologically. Examples are p53, STAT5, Groucho, TEL, Sp1(Clore et al, 1994; Chen et al, 1998; John et al, 1999 Jousset et al,1997) and, as shown here, PML, PLZF and ETO. Regardless of theirimplications for leukemogenesis, the inventors' findings with PML-RARand AML 1-ETO provide genetic evidence to demonstrate thatoligomerization per se has profound effects on the regulatory propertiesof a transcription factor, to the point of radically modifying itsbiological effects. The L\PC-AML1-ETO deletion derivative (that cannotform HMW complexes) is still competent to bind NCoR, but is unable torepress transcription from an AML1 target gene (and to blockdifferentiation). This finding demonstrates that the self-associationdomain of ETO is essential in directing efficient recruitment of theNCoR/HDAC complex and transcriptional repression. ETO is a transcriptionfactor that physiologically forms HMW complexes and recruits theNCoR-HDAC complex. Although the natural targets of ETO are stillunknown, the data presented here predicts that oligomerization iscrucial for the natural function of ETO. Therefore, increased density ofinteracting domains for transcriptional coregulators, owing to formationof oligomeric complexes, may constitute a general mechanism to generatehigh local concentrations of coregulators. Notably, a single pointmutation that prevents STAT5 tetramerization decreases levels ofSTAT5-mediated transcriptional activation (John et al., 1999).

[0152] A corollary derivable from these considerations: to functionefficiently as “catalytic” centres for recruitment of coregulators, theinteractions underlying the formation of the oligomeric complexes mustbe considerably tighter than those responsible for coregulatorrecruitment. The PML-RAR HMW complexes, but not the associations of RARor PML-RAR with NCoR/HDAC, were resistant to strong lysis conditions,such as high salt (2M KCl), detergents (1% Triton or NP-40), reducingagents (50 mM DTT) (unpublished data), suggesting that coiledcoil-mediated interactions are considerably more stable than theRAR-NCoR interactions. Consistently, in the experimental conditions usedfor gel filtration chromatography, the inventors did not find evidenceof RAR-associated coregulators in HMW PML-RAR complexes (RAR itself wasfound to fractionate as a monomer). However, oligomerization andstrength of self-association are not determined only by coiled coilstructures. The p53 tetramerization domain, as compared to the PMLcoiled coil region, conferred similar biochemical, transcriptional andbiological properties upon fusion with RAR. The APL fusion proteinsPLZF-RAR and NPM-RAR formed HMW complexes. However, neither PLZF nor NPMhave predicted coiled coil regions in their sequences. PLZF contains aBTB-POZ domain which, in the context of the GAGA transcription factor,mediates the formation of oligomeric complexes (Katsani et al., 1999);NPM contains an alternative amino-terminal oligomerization domain, thatmediates the formation of hexameric structures (Chan and Chan, 1995).Not tested here, NuMA-RAR, another fusion protein of RAR found in onecase of APL, also contains a strong oligomerization domain in the NuMAmoiety of the fusion protein (Harborth et al., 1999).

[0153] However, the extreme heterogeneity of the protein-proteininteraction modules that are apparently competent for oligomerizationpoints to additional functions of these modules within the HMWcomplexes. The PML coiled coil- and the p53-RAR fusion proteins hadidentical transcriptional repression properties and effects ondifferentiation. However, only the coiled coil-RAR fusion was able tomount a RA response. The PML coiled coil region can also direct theformation of PML/PML-RAR hetero-oligomeric complexes and PML itself hasbeen shown to function as a co-factor in the RA pathway (Wang et al.,1998) and to associate with histone acetylases (Doucas et al., 1999).Therefore, the RA-response that is observed in PML-RAR expressing cellsmight be a consequence of the unique ability of the PML coiled coilregion to recruit wild-type PML proteins to PML-RAR oligomers.

[0154] The leukemia-associated fusion proteins always contain at leastone transcription factor. The present inventors have shown for the firsttime that oligomerization, per se, is sufficient to activate theoncogenic potential of a transcription factor (RAR); that twoleukemia-associated fusion proteins (PML-RAR and AML 1-ETO) exist invivo as oligomeric complexes; and that in both these casesoligomerization is indispensable for oncogenesis. Oligomerization oftranscription factors might, therefore, serve as a general mechanism ofoncogene activation in leukaemias. TEL is a member of the Ets family oftranscription factors, which contains an oligomerization domain and isfound in the leukaemia-associated TEL-AML1 fusion protein. The TELoligomerization domain is conserved in TEL-AML1 and is required for itstranscriptional repressive properties (Jousset et at., 1997; Uchida etal., 1999). Notably, the portion of AML1 retained in this fusionincludes a carboxy-terminal region lost in AML1-ETO and recently shownto recruit the Groucho family of co-repressors, suggesting thatoligomerization might lead, also in this case, to constitutivetranscriptional repressive activity of the fusion protein (Dittmer andNordheim, 1998; Jousset et al., 1997; Levanon et al., 1998; Uchida etal., 1999). The oligomerization domain of TEL is also found in otherleukemia-associated fusion proteins together with tyrosine-kinases(Platelet-derived growth factor receptor β or JAK2). In these cases,however, oligomerization leads to the constitutive activation of theassociated tyrosine kinase (Carroll et al., 1996; Lacronique et al.,1997), a well-characterized and frequent mechanism of oncogeneactivation in human tumours. Therefore, oligomerization appears to be amechanism of oncogene activation for both tyrosine kinases andtranscription factors. Alterations of the oligomerization status offusion proteins, containing either tyrosine kinases or transcriptionfactors, are then expected to affect their oncogenic potential.Interestingly, oligomerization inhibitory peptides are able to revert invitro the transforming phenotype of BCR/ABL, a tyrosine kinase fusionprotein found in chronic myelogenous leukemia (Guo et al., 1998).

[0155] In conclusion, the present inventors have established for thefirst time the mechanism of altered recruitment of the NCoR/HDAC complexby PML-RAR in APL, and presented evidence suggesting thatoligomerization of a transcription factor represents a potentiallywidespread mechanism of transcriptional regulation and oncogenictransformation. They have additionally shown that the approach of fusinga heterologous oligomerization domain (preferably, the coiled coildomain of PML) to a target protein may result paradoxically (given theirobservation that a similar phenomenon occurs in an oncogenic protein) indesirable properties for the said modified “target”, resulting in i)either a target with enhanced functional activity, or ii) in a targetwith impaired function, depending on the properties of the target priormodification. Thus, the inventors have established the theoretical andexperimental basis for an approach that may have several applications inthe biotechnology field, and in the design of novel therapies againstvarious forms of diseases.

[0156] Materials and Methods

[0157] Plasmids

[0158] The following plasmids have been previously described:pSG5-PML-RAR, pSG5-PML-RAR AHT, pSG5-RAR, pSG5-RAR AHT,pSG5-ΔCC-PML-RAR, pSG5-CC-RAR, pGEX-NCoR (1782-2453), pCMV-GAL4-VP16,pcDNA3-PML, pcDNA3-NPM, pcDNA3-PLZF, pcDNA3-PLZF-RAR, pcDNA3-NPM-RAR.pcDNA3-AML1, pcDNA3-ETO, pcDNA3-myc-AML1-ETO(Gelmetti et al., 1998;Grignani et al., 1996; Lillie and Green, 1989; Zhang et al., 1997).pMAL-PML-RAR was obtained by site-directed mutagenesis of the 1^(st) ATGof the PML-RAR cDNA (from pSG5-PML-RAR) and insertion of an EcoRI siteused for in-frame cloning in pMAL-C2 (New England BioLabs).pcDNA3-p53-RAR was cloned by insertion of a PCR fragment containing thetetramerization domain of p53 (Chen et al., 1998; Clore et al., 1994)carrying an optimal Kozak sequence and flanked by the appropriaterestriction sites for in-frame cloning at the ATG of pSG5-RAR.pcDNA3-ΔPC-myc-AML1-ETO, pcDNA3-ΔZF-myc-AML-ETO were obtained byPCR-mediated deletion mutagenesis of the indicated regions ofpcDNA3-AML1-ETO as described in the Results section (Gelmetti et al.,1998; Lutterbach et al., 1998; Lutterbach et al., 1998). PSG5-CC-p53 andpSG5-CC-RAR were obtained by replacing the RAR fragment from thepSG5-CC-RAR construct with a p53 fragment (Pearson et al., 2000), or aTR cDNA, both carrying a mutated ATG for in-frame cloning at the EcoRVsite of pSG5-CC-RAR. The retroviral vectors were cloned by insertion ofthe appropriate cDNAs into the EcoRI site of Pinco (Grignani et al.,1998). pRARE-G5-TATA was obtained by inserting five GAL4 binding sitesand the minimal promoter sequence from pG5E1b into the Xhol-Hindlllsites of the pGL2 plasmid-Promega- (Lillie and Green, 1989).Oligonucleotides containing the RARE from the RARβ2 promoter (Minucci etal., 1994) were inserted at the Mlu1 site. MDR1-luc was obtained by PCRof the MDR1 promoter region (Lutterbach et al., 1998) from a genomicclone and subsequent cloning in pGL2. All of the constructs have beenverified by sequencing.

[0159] Pull-Down Assays and Co-Immunoprecipitation Experiments

[0160] GST-NCoR (1782-2453) or GST-NCoR (RDIII) purification and invitro interaction experiments were performed as described, incubatingthe indicated amounts of GST-NCoR attached to a constant amount ofglutathione-agarose beads in the presence of the appropriate ³⁵-Slabelled, in vitro translated proteins(Gelmetti et al., 1998; Grignaniet al., 1998; Zamir et al., 1996). The input lanes represent 100% of thetotal. Coimmunoprecipitation experiments were performed asdescribed(Gelmetti et al., 1998), using extracts from transientlytransfected COS-1 cells, or in vitro translated products.

[0161] Transactivation Assays

[0162] Transient transfection of HeLa, NIH 3T3, SAOS and C33A cells wasperformed by calcium phosphate as described (Lillie and Green, 1989;Minucci et al., 1994). p53-/- MEFs were transiently transfected bylipofection as described (Pearson et al., 2000). Light units werenormalized to expression of a co-transfected β-galactosidase expressionplasmid. Results are presented as the mean+standard deviations of atleast three independent experiments.

[0163] Gel Filtration Analysis of RA Binding Activity.

[0164] Nuclear extracts (1-1.5 mg) from U937 cells or from transientlytransfected COS-1 cells were incubated with 5 nM [³H]-RA (NEN LifeScience) for 18 hrs at 4° C. [³H]-RA binding was analyzed using a gelfiltration size exclusion column Superose 6 HR 10/30 (Pharmacia,Uppsala, Sweden) equilibrated in column buffer (Hepes 20 mM pH 7.4, EDTA1 mM, DTT 1 mM, aprotinin and leupeptin 10 μg/ml, pepstatin 2 μg/ml, 1mM PMSF, glycerol 1%, NaF 5 mM, KCl 0.4M). The [³H]-RA binding profilewas measured using a Ramona 5 Radioactivity Monitoring radioflowdetector analyser (Ray test, Milano, Italy) using a splitting device,electronically connected with the fraction collector.

[0165] Biochemical Purification of PML-RAR HMW Complexes and SizeExclusion Chromatography (SEC)

[0166] Nuclear extracts from U937 cells stably expressing PML-RAR (PR9clone) were prepared as described (Nervi et al., 1992). Extracts werepartially purified onto a Heparin-Sepharose column and then loaded on aSuperose 6 HR 10/30 gel filtration column equilibrated in column buffer.The PML-RAR-containing fractions were diluted with incubation bufferlacking KCl (Hepes 20 mM pH 7.4, EDTA 1 mM, DTT 1 mM, aprotinin andleupeptin 10 μg/ml, pepstatin 2μg/ml, 1 mM PMSF, glycerol 10%, MgCl₂ 3mM, NaF 5 mM, NP40 0.1%; final 0.1M KCl) and incubated for 1 hour at 4°C. with biotynilated RARE double-stranded oligonucleotide (7 μg/mgstarting material) coupled to streptavidine-agarose beads as described(Blanco et al., 1998; Minucci et al., 1994). As controls, the inventorsused either streptavidine-agarose beads alone, or performed theincubation with RARE-containing beads in the presence of a 100 foldexcess RARE competitor in solution. Beads were eluted in buffercontaining 1M KCl: aliquots of the eluted material (corresponding toapproximately 10% of the PML-RAR amount present in the nuclear extracts)were analyzed by SDS-PAGE followed by silver stain or Western blotting,or were re-loaded onto a Superose 6 gel filtration column and thenanalyzed by Western blotting.

[0167] Expression and Characterization of Recombinant PML-RAR

[0168] pMAL-PML-RAR was expressed in BL21 cells. Bacterial lysates wereincubated with amylose beads for two hours at 4° C. MBP-PML-RAR waseluted by adding maltose (20 mM), loaded onto a MonoQ column (SMARTsystem, Pharmacia Biotech), and then subjected to an additional round ofamylose affinity chromatography. MBP-PML-RAR was eluted and incubatedwith factor Xa to cleave the MBP moiety and yield purified PML-RAR, thatwas subsequently analysed by gel filtration chromatography (Superose 6column, SMART system, Pharmacia Biotech).

[0169] In Vivo Cross/Inking Experiments

[0170] U937 PR9 cells were grown for 1 hour in medium devoid of cysteineand methionine, and then incubated for 8 hours in the presence of ³⁵-Slabelled cysteine and methionine (Amersham). Before harvesting, cellswere incubated for 30 minutes at room temperature in PBS plus 0.1 mMDTBP (Dimethyl 3,3′- dithiobisproprionamidate-2HCl, Pierce). Isolatednuclei were extracted in modified RIPA buffer (150 mM NaCl, 1% NonidetP-40, 1% sodyum deoxycholate, 0.2% SDS, 2 mM EDTA, 5 mM NaF, aprotininand leupeptin 10 μg/ml, pepstatin 2μg/ml, 1 mM PMSF, 100 mM Tris-Cl pH7.4). The extracts were collected and analysed by gel filtrationchromatography on a Superose 6 HR 10/30 column (Pharmacia Biotech)equilibrated and calibrated with globular molecular weight markers(Pharmacia Biotech) in the same buffer used for nuclear extraction. HMWPML-RAR complexes were immunoprecipitated with an anti-PML monoclonalantibody (Flenghi et al., 1995) or an unrelated antibody coupled toProtein G-Sepharose beads. The immunoprecipitated material was elutedfrom the beads in 1% SDS, and an aliquot was further analysed by gelfiltration chromatography (Superose 6, SMART system, Pharmacia Biotech)to verify the integrity of the immunoprecipitated, cross-linked HMWcomplexes.

[0171] In Vitro Differentiation of Murine Hematopoietic Progenitor CellsTransduced with Retroviral Constructs

[0172] Murine hematopoietic progenitors were purified from the bonemarrow of 12 weeks old BALB-C mice using commercially available kits(StemCell Tecnology). Cells were selected on the basis of the absence oflineage differentiation markers (lin−). Purified cells werepre-stimulated for two days in medium containing IL-3 (20 ng/ml), IL-6(20 ng/ml) and stem cell factor (SCF, 100 ng/ml) and then attached toRetronectin (Takara Shuzo)-coated multiwell plates. Cells were incubatedfor 48 hours with the filtered supernatant from ecotropic packagingcells (Phoenix) transiently transfected with the indicated retroviralconstructs (Grignani et al., 1998). Infected cells were sorted by FACSon the basis of their expression of GFP as a selectable marker from thevectors. Cells were seeded in methylcellulose plates (StemCellTecnology) supplemented with IL-3, IL-6, and SCF as above and with theaddition of G-CSF (60 ng/ml) and GM-CSF (20 ng/ml). After 8-10 days,cells were harvested and incubated with biotinylated anti Mac1 or GR1antibodies (Pharmingen), followed by Cytochrome-C Streptavidin(Becton-Dickinson) and FACS analysis to evaluate the extent ofdifferentiation.

[0173] Injection of Xenopus Oocytes and Mobility Shift Assays

[0174] Linearized plasmids were transcribed using a mMessage mMachinekit (Ambion) to produce capped mRNAs. Approximately 30 nl mRNA/Xenopusoocyte cytoplasm were injected as previously described (Minucci et al.,1998). The injected ooxytes were the incubated for 16 h at 18° C. andthe protein expression evaluated by Western blot analysis. Mobilityshift assays were performed in a mixture of 20 μl containing thespecific DNA binding fragment, 0.5 μg of poly(dl:dC) in homogenizationbuffer as described (Landsberger and Wolffe, 1995; Minucci et al.,1998).

[0175] References

[0176] Ahmad, K. F ., et al. (1998). Proc Natl Acad Sci USA 95, 12123-8.

[0177] Benedetti, L., et al. (1997) Blood 90, 1175-85.

[0178] Blanco, J. C., et al (1998). Genes Dev 12, 1638-51.

[0179] Brown, D., et al. (1997). Proc Natl Acad Sci USA 94, 2551-6.

[0180] Carroll, M., et al. (1996). Proc Natl Acad Sci USA 93, 14845-50.

[0181] Chambon, P. (1996). Faseb J. 10,940-54.

[0182] Chakrabarti, S. R., and Nucifora, G. (1999). Biochem Biophys ResCommun 264, 871-7.

[0183] Chan, P. K., and Chan, F. V. (1995). Biochim Biophys Acta 1262,37-42.

[0184] Chang, K. S., et al. (1995). Blood 85, 3646-53.

[0185] Chen, G., et al. (1998). Mol Cell Biol 18, 7259-68.

[0186] Cheng, G. X., et al. (1999). Proc Natl Acad Sci USA 96, 6318-23.

[0187] Clore, G. M., et al. (1994). Science 265, 386-91.

[0188] David, G., et al. (1998). Oncogene 16, 2549-56.

[0189] Dittmer, J., and Nordheim, A. (1998). Biochim Biophys Acta 1377,F1-11.

[0190] Doucas, V., et al. (1999). Proc Natl Acad Sci USA 96, 2627 -32.

[0191] Du, C., et al. (1999). Blood 94, 793-802.

[0192] Flenghi, L., et al. (1995) Blood 85, 1871-80.

[0193] Gelmetti, V., (1998). Mol Cell Biol 18, 7185-91.

[0194] Grignani, F ., et al. (1999). Oncogene 18, 6313-21.

[0195] Grignani, F., et al. (1998). Nature 391, 815-8.

[0196] Grignani, F., et al. (1993). Cell 74, 42331.

[0197] Grignani, F., et al. (1998). Cancer Res 58, 14-9.

[0198] Grignani, F., et al. (1996). Embo J 15, 4949-58.

[0199] Grisolano, J. L., et al. (1997). Blood 89, 376-87.

[0200] Grunstein, M. (1997). Nature 389, 349-352.

[0201] Guidez, F., et al. (1998). Blood 91, 2634-42.

[0202] Guo, X. V., et al. (1998). Oncogene 17, 825-33.

[0203] Harborth, J., et al. (1999). Embo J 18, 1689-700.

[0204] He, L. Z., (1998). Nat Genet 18, 126-35.

[0205] Hirano, T., and Mitchison, T. J. (1994). Cell 79,449-58.

[0206] Horlein, A. J., et al. (1995). Nature 377, 397-404.

[0207] Inoue et al (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11117-11121.

[0208] John, S., et al. (1999). Mol Cell Biol 19, 1910-8.

[0209] Jousset, C., et al. (1997). Embo J 16, 69-82.

[0210] Kadonaga, J. T. (1998). Cell 92, 301-13.

[0211] Kammerer, R. A., et al. (1998). J Biol Chem 273, 10602-8.

[0212] Katsani, K. R., et al. (1999). Embo J 18, 698-708.

[0213] Kitabayashi, I., et al. (1998). Embo J 17, 2994-3004.

[0214] Lacronique, V., et al. (1997). Science 278, 1309-12.

[0215] Landsberger, N., and Wolffe, A. P. (1995). Semin Cell Biol 6,191-9.

[0216] Lavau, C., et al. (1997). Embo J 16, 4226-37.

[0217] Leonhardt et al., Genomics (1994) 19, 130-136.

[0218] Levanon, D., et al. (1998). Proc Natl Acad Sci USA 95, 11590-5.

[0219] Lillie, J. W., and Green, M. R. (1989). Nature 338, 39-44.

[0220] Lin, R. J., et al. (1998). Nature 391, 811-4.

[0221] Look, A. T. (1997). Science 278, 1059-64.

[0222] Lupas, A. (1996). Trends Biochem Sci 21, 375-82.

[0223] Lutterbach, B., et al. (1998). Mol Cell Biol 18, 3604-11.

[0224] Lutterbach, B., et al. (1998). Mol Cell Biol 18, 7176-84.

[0225] Mangelsdorf, D. J., and Evans, R. M. (1995). Cell 83, 841-50.

[0226] McWhirter, J. R., et al. (1993). Mol Cell Biol 13, 7587 -95.

[0227] Melnick, A., and Lrcht, J. D. (1999). Blood 93, 3167-215.

[0228] Meyers, S., et al. (1995). Mol Cell Biol 15, 1974-82.

[0229] Minucci, S., et al. (1994). Mol Cell Biol. 14, 360-72.

[0230] Minucci, S., and Ozato, K. (1996). Curr Opin Genet Dev 6, 567-74.

[0231] Minucci, S., et al. (1998). Mol Endocrinol. 12, 315-24.

[0232] Minucci, S., and Pelicci, P. G. (1999). Semin Cell Dev. Biol. 10,215-25.

[0233] Nervi, C., et al. (1992). Cancer Res 52, 3687-92.

[0234] Okuda, K., et al. (1997). J Clin Invest 100, 1708-15.

[0235] Pazin, M., and Kadonaga, J. T. (1997). Cell 89, 325-328.

[0236] Pearson, M et al. (2000). Nature, 406, 207-210.

[0237] Pereira, D. S., et al. (1998). Proc Natl Acad Sci USA 95,8239-44.

[0238] Ptashne, M., and Gann, A. (1997). Nature 386, 569- 77.

[0239] Purton, L. E., et al. (1999). Blood 94, 483-95.

[0240] Rabbitts, T. H. (1994). Nature 372, 143-9.

[0241] Rabbitts, T. H. (1991). Cell 67, 641-4.

[0242] Redner, R. L., et al. (1999). Blood 94, 417 -28.

[0243] Ruthardt, M., et al. (1997). Mol Cell Biol 17, 4859-69.

[0244] Schwaller, J., et al. (1998). Embo J 17, 5321-33.

[0245] Shivdasani, R. A., and Orkin, S. H. (1996). Blood 87, 4025-39.

[0246] Siegel, L. M., and Monty, K. J. (1966). Biochim Biophys Acta 112,346-62.

[0247] Slany, R. K., et al. (1998). Mol Cell Biol. 18, 122-9.

[0248] Stunnenberg, H. G., et al. (1999). Biochim Biophys Acta 1423,F15-33.

[0249] Saurin et al Trends Biochem Sci, 21, 208-214 (1996)

[0250] Tenen, D. G., et al. (1997). Blood 90, 489-519.

[0251] Tjian, R., and Maniatis, T. (1994). Cell 77, 5-8.

[0252] Uchida, H., et al. (1999). Oncogene 18, 1015-22.

[0253] Wang, J., et al. (1998). Proc Natl Acad Sci USA 95, 10860-5.

[0254] Wang, Z. G., et al. (1998). Science 279, 1547-51.

[0255] Westervelt, P., and Ley, T. J. (1999). Blood 93, 2143-8.

[0256] Wolffe, A. P., et al. (1997). Biochem Soc Trans 25, 612-5.

[0257] Xu, L., et al. (1999). Curr Opin Genet Dev 9, 140-7.

[0258] Zamir, I., et al. (1996). Mol Cell Biol. 16, 5458-65.

[0259] Zhang, V. W., et al. (1997). Mol Cell Biol. 17, 4133-45.

1 15 1 141 PRT Homo sapiens 1 Ser Glu Leu Lys Cys Asp Ile Ser Ala GluIle Gln Gln Arg Gln Glu 1 5 10 15 Glu Leu Asp Ala Met Thr Gln Ala LeuGln Ala Leu Gln Glu Gln Asp 20 25 30 Ser Ala Glu Gly Ala Val His Ala GlnMet His Ala Ala Val Gly Gln 35 40 45 Leu Gly Arg Ala Arg Ala Glu Thr GluGlu Leu Ile Arg Glu Arg Val 50 55 60 Arg Gln Val Val Ala His Val Arg AlaGln Glu Arg Glu Leu Leu Glu 65 70 75 80 Ala Val Asp Ala Arg Tyr Gln ArgAsp Tyr Glu Glu Met Ala Ser Arg 85 90 95 Leu Gly Arg Leu Asp Ala Val LeuGln Arg Ile Arg Thr Gly Ser Ala 100 105 110 Leu Val Gln Arg Met Lys CysTyr Ala Ser Asp Gln Glu Val Leu Asp 115 120 125 Met His Gly Phe Leu ArgGln Ala Leu Cys Arg Leu Arg 130 135 140 2 138 PRT Mus sp. 2 Ser His LeuHis Cys Asp Ile Gly Glu Glu Ile Gln Gln Trp His Glu 1 5 10 15 Glu LeuGly Thr Met Thr Gln Thr Leu Glu Glu Gln Gly Arg Thr Phe 20 25 30 Asp SerAla His Ala Gln Met Cys Ser Ala Ile Gly Gln Leu Asp His 35 40 45 Ala ArgAla Asp Ile Glu Lys Gln Ile Gly Ala Arg Val Arg Gln Val 50 55 60 Val AspTyr Val Gln Ala Gln Glu Arg Glu Leu Leu Glu Ala Val Asn 65 70 75 80 AspArg Tyr Gln Arg Asp Tyr Gln Glu Ile Ala Gly Gln Leu Ser Cys 85 90 95 LeuGlu Ala Val Leu Gln Arg Ile Arg Thr Ser Gly Ala Leu Val Lys 100 105 110Arg Met Lys Leu Tyr Ala Ser Asp Gln Glu Val Leu Asp Met His Ser 115 120125 Phe Leu Arg Lys Ala Leu Cys Ser Leu Arg 130 135 3 132 PRT Homosapiens 3 Phe Gln Glu His Lys Asn His Ser Thr Val Thr Val Glu Glu AlaLys 1 5 10 15 Ala Glu Lys Glu Thr Glu Leu Ser Leu Gln Lys Glu Gln LeuGln Leu 20 25 30 Lys Ile Ile Glu Ile Glu Asp Glu Ala Glu Lys Trp Gln LysGlu Lys 35 40 45 Asp Arg Ile Lys Ser Phe Thr Thr Asn Glu Lys Ala Ile LeuGlu Gln 50 55 60 Asn Phe Arg Asp Leu Val Arg Asp Leu Glu Lys Gln Lys GluGlu Val 65 70 75 80 Arg Ala Ala Leu Glu Gln Arg Glu Gln Asp Ala Val AspGln Val Lys 85 90 95 Val Ile Met Asp Ala Leu Asp Glu Arg Ala Lys Val LeuHis Glu Asp 100 105 110 Lys Gln Thr Arg Glu Gln Leu His Ser Ile Ser AspSer Val Leu Phe 115 120 125 Leu Gln Glu Phe 130 4 126 PRT Homo sapiens 4Arg Asp Cys Gln Leu Leu Glu His Lys Glu His Arg Tyr Gln Phe Leu 1 5 1015 Glu Glu Ala Phe Gln Asn Gln Lys Gly Ala Ile Glu Asn Leu Leu Ala 20 2530 Lys Leu Leu Glu Lys Lys Asn Tyr Val His Phe Ala Ala Thr Gln Val 35 4045 Gln Asn Arg Ile Lys Glu Val Asn Glu Thr Asn Lys Arg Val Glu Gln 50 5560 Glu Ile Lys Val Ala Ile Phe Thr Leu Ile Asn Glu Ile Asn Lys Lys 65 7075 80 Gly Lys Ser Leu Leu Gln Gln Leu Glu Asn Val Thr Lys Glu Arg Gln 8590 95 Met Lys Leu Leu Gln Gln Gln Asn Asp Ile Thr Gly Leu Ser Arg Gln100 105 110 Val Lys His Val Met Asn Phe Thr Asn Trp Ala Ile Ala Ser 115120 125 5 146 PRT Homo sapiens 5 Gly Arg His Arg Asp His Gln Val Ala AlaLeu Ser Glu Arg Tyr Asp 1 5 10 15 Lys Leu Lys Gln Asn Leu Glu Ser AsnLeu Thr Asn Leu Ile Lys Arg 20 25 30 Asn Thr Glu Leu Glu Thr Leu Leu AlaLys Leu Ile Gln Thr Cys Gln 35 40 45 His Val Glu Val Asn Ala Ser Arg GlnGlu Ala Lys Leu Thr Glu Glu 50 55 60 Cys Asp Leu Leu Ile Glu Ile Ile GlnGln Arg Arg Gln Ile Ile Gly 65 70 75 80 Thr Lys Ile Lys Glu Gly Lys ValMet Arg Leu Arg Lys Leu Ala Gln 85 90 95 Gln Ile Ala Asn Cys Lys Gln CysIle Glu Arg Ser Ala Ser Leu Ile 100 105 110 Ser Gln Ala Glu His Ser LeuLys Glu Asn Asp His Ala Arg Phe Leu 115 120 125 Gln Thr Ala Lys Asn IleThr Glu Arg Val Ser Met Ala Thr Ala Ser 130 135 140 Ser Gln 145 6 144PRT Homo sapiens 6 Asp His Gln Val Ala Ser Leu Asn Asp Arg Phe Glu LysLeu Lys Gln 1 5 10 15 Thr Leu Glu Met Asn Leu Thr Asn Leu Val Lys ArgAsn Ser Glu Leu 20 25 30 Glu Asn Gln Met Ala Lys Leu Ile Gln Ile Cys GlnGln Val Glu Val 35 40 45 Asn Thr Ala Met His Glu Ala Lys Leu Met Glu GluCys Asp Glu Leu 50 55 60 Val Glu Ile Ile Gln Gln Arg Lys Gln Met Ile AlaVal Lys Ile Lys 65 70 75 80 Glu Thr Lys Val Met Lys Leu Arg Lys Leu AlaGln Gln Val Ala Asn 85 90 95 Cys Arg Gln Cys Leu Glu Arg Ser Thr Val LeuIle Asn Gln Ala Glu 100 105 110 His Ile Leu Lys Glu Asn Asp Gln Ala ArgPhe Leu Gln Ser Ala Lys 115 120 125 Asn Ile Ala Glu Arg Val Ala Met AlaThr Ala Ser Ser Gln Val Leu 130 135 140 7 67 PRT Mus sp. 7 Cys Gln LeuAsn Ala His Lys Asp His Gln Tyr Gln Phe Leu Glu Asp 1 5 10 15 Ala ValArg Asn Gln Arg Lys Leu Leu Ala Ser Leu Val Lys Arg Leu 20 25 30 Gly AspLys His Ala Thr Leu Gln Lys Asn Thr Lys Glu Val Arg Ser 35 40 45 Ser IleArg Gln Val Ser Asp Val Gln Lys Arg Val Gln Val Asp Val 50 55 60 Lys MetAla 65 8 164 PRT Homo sapiens 8 Asp Leu Glu Ala Thr Leu Arg His Lys LeuThr Val Met Tyr Ser Gln 1 5 10 15 Ile Asn Gly Ala Ser Arg Ala Leu AspAsp Val Arg Asn Arg Gln Gln 20 25 30 Asp Val Arg Met Thr Ala Asn Arg LysVal Glu Gln Leu Gln Gln Glu 35 40 45 Tyr Thr Glu Met Lys Ala Leu Leu AspAla Ser Glu Thr Thr Ser Thr 50 55 60 Arg Lys Ile Lys Glu Glu Glu Lys ArgVal Asn Ser Lys Phe Asp Thr 65 70 75 80 Ile Tyr Gln Ile Leu Leu Lys LysLys Ser Glu Ile Gln Thr Leu Lys 85 90 95 Glu Glu Ile Glu Gln Ser Leu ThrLys Arg Asp Glu Phe Glu Phe Leu 100 105 110 Glu Lys Ala Ser Lys Leu ArgGly Ile Ser Thr Lys Pro Val Tyr Ile 115 120 125 Pro Glu Val Glu Leu AsnHis Lys Leu Ile Lys Gly Ile His Gln Ser 130 135 140 Thr Ile Asp Leu LysAsn Glu Leu Lys Gln Cys Ile Gly Arg Leu Gln 145 150 155 160 Glu Leu ThrPro 9 130 PRT Mus sp. 9 Leu Ser Gln Ala Ser Ala Asp Leu Glu Tyr Lys LeuArg Asn Lys Leu 1 5 10 15 Thr Ile Met His Ser His Ile Asn Gly Ala ThrLys Ala Leu Glu Asp 20 25 30 Val Arg Ser Lys Gln Gln Cys Val Gln Asp SerMet Lys Arg Lys Met 35 40 45 Glu Gln Leu Arg Gln Glu Tyr Met Glu Met LysAla Val Ile Asp Ala 50 55 60 Ala Glu Thr Ser Ser Leu Arg Arg Leu Lys GluGlu Glu Lys Arg Val 65 70 75 80 Tyr Gly Lys Phe Asp Thr Ile Tyr Gln ValLeu Val Lys Lys Lys Ser 85 90 95 Glu Met Gln Lys Leu Lys Ala Glu Val GluLeu Ile Met Asp Lys Gly 100 105 110 Asp Glu Phe Glu Phe Leu Glu Lys AlaAla Lys Leu Gln Gly Glu Ser 115 120 125 Thr Lys 130 10 106 PRT Xenopuslaevis 10 Glu Ala Ser Leu Lys Val Thr Glu Gln Leu Ser Ser Glu Gln SerAsp 1 5 10 15 Lys Ile Glu Gln His Asn Lys Asn Met Ser Gln Tyr Lys GluHis Ile 20 25 30 Thr Ser Glu Phe Glu Lys Leu His Lys Phe Leu Arg Glu ArgGlu Glu 35 40 45 Lys Leu Leu Glu Gln Leu Lys Glu Gln Gly Glu Asn Leu LeuThr Glu 50 55 60 Met Glu Asn Asn Leu Val Lys Met Gln Glu Ser Gln Asp AlaIle Lys 65 70 75 80 Lys Thr Ile Ser Leu Ala Lys Glu Arg Met Glu Asp ThrAsp Ser Ile 85 90 95 Ser Phe Leu Met Asp Ile Lys Ala Phe Ile 100 105 11121 PRT Homo sapiens 11 Phe Arg Ile Asn Glu Val Val Lys Glu Cys Gln GluLys Leu Gln Val 1 5 10 15 Ala Leu Gln Arg Leu Ile Lys Glu Asp Gln GluAla Glu Lys Leu Glu 20 25 30 Asp Asp Ile Arg Gln Glu Arg Thr Ala Trp LysIle Glu Arg Gln Lys 35 40 45 Ile Leu Lys Gly Phe Asn Glu Met Arg Val IleLeu Asp Asn Glu Glu 50 55 60 Gln Arg Glu Leu Gln Lys Leu Glu Glu Gly GluVal Asn Val Leu Asp 65 70 75 80 Asn Leu Ala Ala Ala Thr Asp Gln Leu ValGln Gln Arg Gln Asp Ala 85 90 95 Ser Thr Leu Ile Ser Asp Leu Gln Arg ArgLeu Thr Gly Ser Ser Val 100 105 110 Glu Met Leu Gln Asp Val Ile Asp Val115 120 12 136 PRT Mus sp. 12 Leu Cys Glu Arg Ser Gln Glu His Arg GlyHis Gln Thr Ala Leu Ile 1 5 10 15 Glu Glu Val Asp Gln Glu Tyr Lys GluLys Leu Gln Gly Ala Leu Trp 20 25 30 Lys Leu Met Lys Lys Ala Lys Ile CysAsp Glu Trp Gln Asp Asp Leu 35 40 45 Gln Leu Gln Arg Val Asp Trp Glu AsnGln Ile Gln Ile Asn Val Glu 50 55 60 Asn Val Gln Arg Gln Phe Lys Gly LeuArg Asp Leu Leu Asp Ser Lys 65 70 75 80 Glu Asn Glu Glu Leu Gln Lys LeuLys Lys Glu Lys Lys Glu Val Met 85 90 95 Glu Lys Leu Glu Glu Ser Glu AsnGlu Leu Glu Asp Gln Thr Glu Leu 100 105 110 Val Arg Asp Leu Ile Ser AspVal Glu His His Leu Glu Leu Ser Thr 115 120 125 Leu Glu Met Leu Gln GlyAla Asn 130 135 13 125 PRT Homo sapiens 13 Leu Glu Glu Ala Ala Gln GluTyr Gln Glu Lys Leu Gln Val Ala Leu 1 5 10 15 Gly Glu Leu Arg Arg LysGln Glu Leu Ala Glu Lys Leu Glu Val Glu 20 25 30 Ile Ala Ile Lys Arg AlaAsp Trp Lys Lys Thr Val Glu Thr Gln Lys 35 40 45 Ser Arg Ile His Ala GluPhe Val Gln Gln Lys Asn Phe Leu Val Glu 50 55 60 Glu Glu Gln Arg Gln LeuGln Glu Leu Glu Lys Asp Glu Arg Glu Gln 65 70 75 80 Leu Arg Ile Leu GlyGlu Lys Glu Ala Lys Leu Ala Gln Gln Ser Gln 85 90 95 Ala Leu Gln Glu LeuIle Ser Glu Leu Asp Arg Arg Cys His Ser Ser 100 105 110 Ala Leu Glu LeuLeu Gln Glu Val Ile Ile Val Leu Glu 115 120 125 14 65 PRT Homo sapiens14 Glu Thr Trp Arg Arg Gly Asp Ala Leu Ser Arg Leu Asp Thr Leu Glu 1 510 15 Thr Ser Lys Arg Lys Ser Leu Gln Leu Leu Thr Lys Asp Ser Asp Lys 2025 30 Val Lys Glu Phe Phe Glu Lys Leu Gln His Thr Leu Asp Gln Lys Lys 3540 45 Asn Glu Ile Leu Ser Asp Phe Glu Thr Met Lys Leu Ala Val Met Gln 5055 60 Ala 65 15 120 PRT Homo sapiens 15 Glu His Arg Glu His Gly Thr ValLeu Leu Arg Asp Val Val Glu Gln 1 5 10 15 His Lys Ala Ala Leu Gln ArgGln Leu Glu Ala Val Arg Gly Arg Leu 20 25 30 Pro Gln Leu Ser Ala Ala IleAla Leu Val Gly Gly Ile Ser Gln Gln 35 40 45 Leu Gln Glu Arg Lys Ala GluAla Leu Ala Gln Ile Ser Ala Ala Phe 50 55 60 Glu Asp Leu Glu Gln Ala LeuGln Gln Arg Lys Gln Ala Leu Val Ser 65 70 75 80 Asp Leu Glu Thr Ile CysGly Ala Lys Gln Lys Val Leu Gln Thr Gln 85 90 95 Leu Asp Thr Leu Arg GlnGly Gln Glu His Ile Gly Ser Ser Cys Ser 100 105 110 Phe Ala Glu Gln AlaLeu Arg Leu 115 120

1. A method of screening for a substance having the ability to modulatethe oligomerization domain of an oligomeric factor such that strongself-association of the oligomeric factors to form oligomeric complexesis prevented or reduced, said method comprising the steps of (a)bringing into contact a first oligomeric factor or the functionalself-association part thereof, a second oligomeric factor or thefunctional self association part thereof, and a test substance, underconditions wherein, in the absence of said test substance, being aninhibitor of association of said oligomeric factors, said oligomericfactors or functional self associating parts thereof interact or bind;and (b) determining the interaction or binding between said oligomericfactors or functional self association parts thereof.
 2. A method ofscreening for a test compound able to bind an oligomerization domain ofan oligomeric factor, said method comprising the steps of (a) bringinginto contact a substance which includes an oligomerization domain whichallows self-association of the oligomeric factors, or a variant,derivative or analogue thereof, and a test compound, and; (b)determining binding between said oligomerization domain and the testcompound.
 3. A method according to claim 1 or claim 2 wherein theoligomeric factor is a fusion protein comprising at least onetranscription factor.
 4. A method according to claim 3 wherein theoligomeric factor is PML-RAR or AML1-ETO.
 5. A method according to claim1 further comprising the steps of isolating said test substance andmanufacturing a medicament comprising the isolated test substance foruse in treating a disease associated with the formation of HMW complexesof oligomeric factors.
 6. A method according to claim 2 furthercomprising the steps of isolating said test compound and manufacturing amedicament comprising the isolated test compound for use in treating adisease associated with the formation of HMW complexes of oligomericfactors.
 7. A method according to claim 5 or claim 6 wherein the diseaseis cancer.
 8. A method according to claim 6 wherein the test compound isan antibody binding domain.
 9. A method of increasing the activity of amonomeric polypeptide in a sample, comprising the steps of producing achimeric protein comprising the polypeptide and an oligomerizationdomain, and adding said chimeric protein to the sample comprisingmonomeric polypeptides thereby allowing self-association of themonomeric polypeptides to the chimeric protein and increasing theactivity of the polypeptide in the sample.
 10. A method according toclaim 9 wherein the chimeric protein is a fusion protein comprising saidpolypeptide and an oligomerization domain.
 11. A method according toclaim 9 or claim 10 wherein the oligomerization domain is the coiledcoil domain of PML.
 12. A method according to claim 9 or claim 10wherein the oligomerization domain is derived from p53, PLZF, NPM orETO.
 13. A method according to any one of claims 9 to 12 wherein thepopulation of monomeric polypeptides in intracellular.
 14. A method ofreducing the activity of an oligomeric polypeptide, comprising the stepsof producing a modified oligomeric polypeptide comprising saidpolypeptide and an additional oligomerization domain, and contactingsaid modified oligomeric polypeptide with a population of oligomericpolypeptides in a sample thereby allowing association of the oligomericpolypeptides to the modified oligomeric polypeptide and as a resultdecreasing the activity of the oligomeric polypeptide in the sample. 15.A method according to claim 14 wherein the modified oligomericpolypeptide is a fusion protein comprising said oligomeric polypeptideand an oligomerization domain.
 16. A method according to claim 14 orclaim 15 wherein the oligomerization domain is the coiled coil domain ofPML.
 17. A method according to any one of claims 14 to 16 wherein theoligomeric polypeptide is p53, cytokines, interleukins, or TNF.
 18. Useof a factor capable of disrupting the activity or formation of HMWcomplexes in the preparation of a medicament for treating a diseaseassociated with the formation of HMW complexes comprising oligomericfactors.
 19. Use according to claim 18 wherein the oligomeric factorsare chimeric transcription factors.
 20. Use according to claim 19wherein the factor is a binding member capable of specifically bindingto the oligomerization domain of the chimeric transcription factor. 21.Use according to claim 19 or claim 20 wherein the oligomerization domainis a coiled coil domain.
 22. Use according to any one of claims 19 to 21wherein the disease is cancer, particularly leukaemia.
 23. Use accordingto any one of claim 19 to 22 wherein the chimeric transcription factoris PML-RAR or AML1-ETO.
 24. Use according to any of claims 19 to 23wherein the binding member is a peptide comprising a coiled coil domainof the chimeric transcription factor.
 25. Use according to claim 24wherein the coiled coil domain has an amino acid sequence having atleast 70% homology with the sequence identified in SEQ ID No.
 1. 26. Useaccording to claim 25 wherein the coiled coil domain has an amino acidsequence having the sequence as shown in SEQ ID No.
 1. 27. A method ofdetermining the presence or absence of a HMW complex comprising two ormore oligomeric factors, said method comprising the steps of obtaining abiological sample from a patient and detecting the presence or absenceof said HMW complex using a specific binding member capable ofspecifically binding to said HMW complex.
 28. A method according toclaim 27 further comprising the step of determining the molecular weightof HMW complex detected in the biological sample.
 29. A method accordingto claim 27 or claim 28 wherein the HMW comprises chimeric transcriptionfactors.
 30. A method according to claim 29 wherein the chimerictranscription factors. comprise PML-RAR or AML1-ETO.
 31. A methodtreating a patient having, or suspected of having, a disease associatedwith the formation of HMW complexes comprising two or more factorscapable of forming self-associating oligomers, said method comprisingthe steps of administering to said patient a substance capable ofpreventing and/or disrupting the activity or formation of said HMWcomplexes.
 32. A method according to claim 31 wherein said substance isa binding member capable of specifically binding to the oligomerizationdomain of the oligomeric factor.
 33. A method according to claim 31 orclaim 32 wherein the oligomeric factors are chimeric transcriptionfactors.
 34. A method according to claim 33 wherein the chimerictranscription factor is PML-RAR or AML1-ETO.
 35. A method according toany one of claims 31 to 34 wherein the disease is cancer.
 36. A methodaccording to claim 35 wherein the disease is leukaemia.
 37. A compoundfor use in modulating the activity of a polypeptide, said compoundcomprising said polypeptide fused to an oligomerization domain of anoligomeric protein.
 38. A compound according to claim 37 wherein theoligomerization domain is the coiled coil domain and the oligomericprotein is PML.
 39. A compound according to claim 37 or claim 38 whereinthe polypeptide is a monomeric polypeptide and the activity of saidpolypeptide is increased.
 40. A compound according to claim 37 or claim38 wherein the polypeptide is oligomeric in nature and the activity ofthe polypeptide is reduced.
 41. A pharmaceutical composition comprisinga compound according to any one of claims 37 to 40 and apharmaceutically acceptable recipient.